Fungal cell wall synthesis gene

ABSTRACT

A reporter system reflecting the transport process that transports GPI-anchored proteins to the cell wall was constructed and compounds inhibiting this process were discovered. Further, genes conferring resistance to the above compounds were identified and methods of screening for compounds that inhibit the activity of the proteins encoded by these genes were developed.  
     Therefore, through the novel compounds, the present invention showed that antifungal agents having a novel mechanism, i.e. inhibiting the process that transports GPI-anchored proteins to the cell wall, could be achieved.

TECHNICAL FIELD

[0001] The present invention relates to DNAs encoding proteinsparticipating in fungal cell wall synthesis, proteins encoded by theDNAs, methods for examining whether or not a certain compound has aninfluence on the transport process involved in the transport ofGPI-anchored proteins to the cell wall, and antifungal agents having aninfluence on the transport process involved in the transport ofGPI-anchored proteins to the cell wall.

BACKGROUND ART

[0002] In recent years, management of opportunistic infections aregaining importance more than ever due to an increase in the number ofelderly people and immunocompromised patients as a result of advancedchemotherapies, etc. Deep-seated mycosis due to Candida, Aspergillus,Cryptococcus, and such, account for a portion of such opportunisticinfections, and the proportion is increasing year after year. The factthat opportunistic infections by many avirulent bacteria occur one afteranother, shows that the problem of infectious diseases will not end aslong as there are underlying diseases that diminish the immune functionsof patients. Although new strategies for infectious diseases control,including the problem of resistant bacteria, will be one of the crucialissues in the soon-to-come aged society, extremely few effectivetherapeutic agents exist at present.

[0003] Up to now, therapeutic agents for fungal infections weredeveloped based mainly on the strategy of creating novel compounds bychemically modifying known structure. However, due to problems such asthe emergence of resistant bacteria, the development of new drugs basedon new mechanisms is eagerly anticipated.

[0004] Considering such circumstances, the inventors focused on a novelapproach in the area of antifungal agents in which the variety oftherapeutic agents is still insufficient. Namely, the present inventorsconcentrated on influencing the onset, progress, and persistence ofinfections by preventing pathogens from showing pathogenicity. In orderto avoid the establishment and progress of infection, the inventorsthought that the most effective way would be to inhibit the adhesiononto the host, which is the first step in the establishment ofinfection, and the subsequent progression of colonization. In addition,a new unprecedented approach, namely, the inhibition of the expressionof adhesion factors themselves, was also carried out.

[0005] In order to inhibit the expression of adhesion factors, thepresent inventors directed their attention to the hypothesis that cellwall glycoproteins such as adhesion factors are first GPI(Glycosylphosphatidylinositol)-anchored to the cell membrane, and thentransported to the cell wall (FIG. 1). To date, 30 or more cell wallglycoproteins including adhesion ligands have been found to betransported via GPI-anchoring (referred to as GPI-anchored proteins).Hence, it was thought that if this transport step is inhibited, it maybe quite possible to inhibit the expression of adhesion factors andmajor cell wall-constituting proteins at the cell wall (Hamada K et al,Mol. Gen. Genet., 258: 53-59, 1998). GPI-anchored proteins have beenreported to be present in Candida, which is a pathogenic fungi (KapteynJ C et al, Eur. J. Cell Biol., 65:402-407, 1994).

[0006] The inventors initiated their research believing that novelantifungal agents that inhibit cell wall synthesis can be produced byinhibiting the process that transports GPI-anchored proteins existing inthe cell membrane of a fungus to the cell wall.

DISCLOSURE OF THE INVENTION

[0007] An objective of this invention is to develop antifungal agentsshowing effects against the onset, progress, and persistence ofinfections by inhibiting the expression of cell wall glycoproteins,inhibiting the cell wall assembly and also adhesion onto cells, andpreventing pathogens from showing pathogenicity.

[0008] In order to screen for compounds that inhibit the process thattransports GPI-anchored proteins to the cell wall, the present inventorsproduced a reporter system that uses a fusion protein comprising areporter enzyme and a transport signal existing in the C-terminus of oneof the GPI-anchored proteins, CWP2 (Van Der Vaat J M et al, J.Bacteriol., 177:3104-3110,1995).

[0009] When a DNA comprising a secretion signal gene + reporter enzymegene + CWP2 C-terminus gene (present or absent) was constructed, and thefusion protein was expressed in Saccharomyces cerevisiae (hereinafter,referred to as S. cerevisiae), it was demonstrated that activity of thereporter enzyme is detected in the cell wall when the CWP2 C-terminus ispresent, and in the culture supernatant when the CWP2 C-terminus isabsent. Accordingly, it was predicted that if the process thattransports GPI-anchored proteins to the cell wall is inhibited by a testsample, the activity of the reporter enzyme in the cell wall will bediminished, or the activity of the reporter enzyme will be found in theculture supernatant. Thus was initiated the screening for compounds thatinhibit the process that transports GPI-anchored proteins to the cellwall using this reporter system.

[0010] From the screening using this reporter system, several compoundsthat inhibit the process that transports GPI-anchored proteins to thecell wall were discovered. A representative example is the compoundshown in formula (Ia).

[0011] The compound shown in the aforementioned formula (Ia)(hereinafter abbreviated as “compound (Ia)”) inhibits the growth of S.cerevisiae and Candida albicans (hereinafter, referred to as C.albicans), and C. albicans cultured in the presence of theaforementioned compound (Ia) shows a weak ability to adhere onto cells.Thus, the aforementioned compound (Ia) was confirmed to suit the initialobjectives of the invention, which was to find a compound that inhibitsthe adhesion of fungi, due to suppressing the expression of the fungaladhesins, based on the inhibition of transport system of GPI-anchoredproteins to the cell wall. Furthermore, observations using atransmission electron microscope confirmed that C. albicans cultured inthe presence of the aforementioned compound (Ia) has an abnormality inits cell wall synthesis.

[0012] Using the aforementioned compound (Ia), the present inventorsproved that antifungal agents based on the mechanism that inhibits theprocess that transports GPI-anchored proteins to the cell wall, could beachieved.

[0013] Furthermore, to specify the target protein on which theaforementioned compound (Ia) acts, the present inventors searched forgenes that confer resistance to the aforementioned compound (Ia).

[0014] A plasmid library of the S. cerevisiae gene was introduced intoS. cerevisiae, and by overexpression, plasmids were collected thatshowed resistance to the abovementioned compound (Ia) The resistant genewas then cloned, the nucleotide sequence was determined, and the genewas named GWT1 (SEQ ID NO: 1). In S. cerevisiae overexpressing the GWT1gene product, the aforementioned reporter enzyme that has the C-terminusof a GPI-anchored protein was transported to the cell wall, even in thepresence of the aforementioned compound (Ia). Furthermore, observationsunder a transmission electron microscope confirmed that the cell wall isnormal even in the presence of the aforementioned compound (Ia).

[0015] Moreover, when point mutations were randomly introduced to thegenomic DNA of S. cerevisiae, and mutant strains R1 and R5 showingspecific resistance to the aforementioned compound (Ia) were isolated,point mutations involving changes of the 405th codon of the GWT1 genefrom GTC to ATC in the R1 mutant strain, and the 140th codon from GGG toAGG in the R5 mutant strain were discovered. Since resistance to theaforementioned compound (Ia) was seen when these mutant GWT1 genes wereintroduced to a GWT1 gene-disrupted strain, resistance to this compoundwas found to be explainable by the GWT1 gene alone. Therefore, thissuggested that the aforementioned compound (Ia) directly acts on theGWT1 gene product to inhibit the function of the GWT1 protein.

[0016] By similar methods, the resistant genes of C. albicans (SEQ IDNOS: 3 and 5) were cloned, the nucleotide sequences were determined, andthe genes were named CaGWT1.

[0017] Furthermore, a database homology search using GWT1, revealed ahomologue (SEQ ID NO: 27) of Schizosaccharomyces pombe (hereinafter,referred to as S. pombe). Furthermore, PCR with primers based on thesequence of the highly conserved region in the proteins encoded by theGWT1 genes of S. cerevisiae, S. pombe, and C. albicans, yieldedhomologues (SEQ ID NOS: 39 and 41) of Aspergillus fumigatus(hereinafter, referred to as A. fumigatus). Furthermore, by performingPCR based on the sequence discovered from a database homology searchwith GWT1, revealed homologues (SEQ ID NOS: 54 and 58) of Cryptococcusneoformans (hereinafter, referred to as C. neoformans).

[0018] More specifically, this invention relates to the following.

[0019] 1. A DNA that encodes a protein having an activity to conferresistance to the compound shown in formula (Ia) on a fungus when theDNA is overexpressed in the fungus, wherein the DNA is selected from thegroup consisting of:

[0020] (a) A DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 2, 4, 6, 28, 40, or 59.

[0021] (b) A DNA comprising the nucleotide sequence of SEQ ID NO: 1, 3,5, 27, 39, 41, 54, or 58.

[0022] (c) A DNA that hybridizes under stringent conditions to a DNAcomprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 27, 39, 41,54, or 58.

[0023] (d) A DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 2, 4, 6, 28, 40, or 59, wherein one or more amino acidshave been added, deleted, substituted, and/or inserted.

[0024] (e) A DNA that is amplified using SEQ ID NOS: 29 and 31 or SEQ IDNOS: 29 and 30 as primers.

[0025] 2. A DNA that encodes a protein having an activity to decreasethe amount of a GPI-anchored protein in the cell wall of a fungus due toa defect in the function of the DNA, wherein the DNA is selected fromthe group consisting of:

[0026] (a) A DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 2, 4, 6, 28, 40, or 59,

[0027] (b) A DNA comprising the nucleotide sequence of SEQ ID NO: 1, 3,5, 27, 39, 41, 54, or 58,

[0028] (c) A DNA that hybridizes under stringent conditions to a DNAcomprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 27, 39, 41,54, or 58,

[0029] (d) A DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 2, 4, 6, 28, 40, or 59, wherein one or more amino acidshave been added, deleted, substituted, and/or inserted, and

[0030] (e) A DNA that is amplified using SEQ ID NOS: 29 and 31 or SEQ IDNOS: 29 and 30 as primers,

[0031] and wherein, “stringent conditions” refer to: for example,hybridization in 4×SSC at 65° C., then washing in 0.1×SSC for 1 hour at65° C.; or in a different method, “stringent conditions” are 4×SSC at42° C. in 50% formamide; or, hybridization in PerfectHyb™ (TOYOBO)solution for 2.5 hours at 65° C., then washing in (i) 2×SSC, 0.05% SDSsolution at 25° C. for 5 minutes, (ii) 2×SSC, 0.05% SDS solution at 25°C. for 15 minutes, and (iii) 0.1×SSC, 0.1% SDS solution at 50° C. for 20minutes;

[0032] a “defect in the DNA function” can occur, when the functionalgene product of the DNA is not expressed or when the expression isdiminished, for example by inserting a DNA that is irrelevant to thecoding region of the DNA, for example a selection marker, using thehomologous recombination technique;

[0033] and a decrease in the protein derived from the GPI-anchoredprotein in the fungal cell wall is quantified by using any one of thefollowing methods alone or in combination: (i) a reporter systemreflecting the process that transports GPI-anchored proteins to the cellwall, (ii) an ELISA that quantifies a GPI-anchored protein in the cellwall, (iii) measuring the activity of a GPI-anchored protein, such asadhesion onto animal cells, or (4) observing the flocculent, fibrousstructure of the outermost layer of the fungal cell by a transmissionelectron microscope.

[0034] 3. A protein encoded by the DNA of 1 or 2.

[0035] 4. A vector into which the DNA of 1 or 2 has been inserted.

[0036] 5. A transformant harboring the DNA of 1 or 2, or the vector of4.

[0037] 6. The transformant of 5 which is a fungus that overexpresses theprotein of 3.

[0038] 7. A fungus, wherein the function of the protein of 3 isdefective.

[0039] 8. A method for producing the protein of 3, which comprises thesteps of culturing the transformant of 5, and collecting the expressedprotein from the transformant, or from the culture supernatant thereof.

[0040] 9. An antibody that binds to the protein of 3.

[0041] 10. A method of screening for a compound having an antifungalaction, wherein the method comprises the steps of:

[0042] (a) contacting a test sample with the protein of 3;

[0043] (b) detecting the binding activity between the protein and thetest sample; and

[0044] (c) selecting a compound having an activity to bind to theprotein.

[0045] 11. A method of screening for a compound that has an antifungalaction, which comprises the steps of:

[0046] (a) contacting a test sample with a fungus that is overexpressingthe protein of 3;

[0047] (b) detecting the amount of transport of a GPI-anchored proteinto the cell wall in the fungus; and

[0048] (c) selecting a compound that diminishes the amount of transportof the GPI-anchored protein to the cell wall detected in step (b) ascompared to the amount of transport detected when the test sample wascontacted with a fungus that is not overexpressing the protein of 3,

[0049] wherein, a decrease in the amount of GPI-anchored proteintransported to the cell wall that results due to the test sample can bedetected, for example, by detecting a decrease in growth rate, swelling,or temperature sensitivity of the cell, or by detecting a decrease ofthe protein derived from the GPI-anchored protein in the cell wall, butpreferably, by detecting a decrease in the protein derived from theGPI-anchored protein at the cell wall;

[0050] and wherein a decrease of the protein derived from theGPI-anchored protein is quantified by using any one of the followingmethods alone or in combination: (i) a reporter system reflecting theprocess that transports GPI-anchored proteins to the cell wall, (ii) anELISA that quantifies one type of the GPI-anchored protein in the cellwall, (iii) measuring the activity of a GPI-anchored protein such asadhesion to animal cells, and (iv) observing the flocculent, fibrousstructure of the outermost layer of a fungal cell by a transmissionelectron microscope.

[0051] 12. A compound having an antifungal action that is isolated bythe screening of 10 or 11.

[0052] 13. An antifungal agent, comprising as an active ingredient acompound that inhibits the transport of GPI-anchored proteins to thecell wall of a fungus.

[0053] 14. An antifungal agent, comprising as an active ingredient theantibody of 9 or the compound of 12.

[0054] 15. The antifungal agent of 13, comprising as an activeingredient the compound represented by the general formula (I), a saltthereof, or a hydrate thereof, wherein in formula (I):

[0055] [R^(1a) and R^(2a) are identical to or different from each otherand denote individually a hydrogen atom, halogen atom, hydroxyl group,nitro group, cyano group, trifluoromethyl group, trifluoromethoxy group,a substituted or unsubstituted C₁₋₆ alkyl group, C₂₋₆ alkenyl group,C₂₋₆ alkynyl group, a substituted or unsubstituted C₁₋₆ alkoxy group, ora group represented by the formula:

[0056] (wherein X¹ stands for a single bond, carbonyl group, or a grouprepresented by the formula —S(O)₂—;

[0057] R^(5a) and R^(6a) are identical to or different from each otherand denote a hydrogen atom or a substituted or unsubstituted C₁₋₆ alkylgroup). Furthermore, R^(1a) and R^(2a) may together form a condensedring selected from the group consisting of a substituted orunsubstituted benzene ring, a substituted or unsubstituted pyridinering, a substituted or unsubstituted pyrrole ring, a substituted orunsubstituted thiophene ring, a substituted or unsubstituted furan ring,a substituted or unsubstituted pyridazine ring, a substituted orunsubstituted pyrimidine ring, a substituted or unsubstituted pyrazinering, a substituted or unsubstituted imidazole ring, a substituted orunsubstituted oxazole ring, a substituted or unsubstituted thiazolering, a substituted or unsubstituted pyrazole ring, a substituted orunsubstituted isoxazole ring, a substituted or unsubstituted isothiazolering, a substituted or unsubstituted cyclohexane ring, and a substitutedor unsubstituted cyclopentane ring;

[0058] R^(3a) and R^(4a) are identical to or different from each otherand denote individually a hydrogen atom, halogen atom, hydroxyl group,nitro group, cyano group, carboxyl group, formyl group, hydroxyiminogroup, trifluoromethyl group, trifluoromethoxy group, C₁₋₆ alkyl group,C₁₋₆ alkoxy group, C₂₋₆ alkenyl group, C₂₋₆ alkynyl group, a grouprepresented by the formula —C(O)NR^(7a)R^(7b) (wherein R^(7a) and R^(7b)are identical to or different from each other and denote individually ahydrogen atom, or a C₁₋₆ alkyl group), the formula —CO₂R^(7a) (whereinR^(7a) has the same meaning as defined above), the formula—S(O)_(n)R^(7a) (wherein n stands for an integer of 0 to 2 and R^(7a)has the same meaning as defined above), the formula —S(O)₂NR^(7a)R^(7b)(wherein R^(7a) and R^(7b) have the same meaning as defined above), agroup of the formula

[0059] (wherein X² denotes a single bond, carbonyl group, or a group ofthe formula —S(O)₂—;

[0060] R^(5b) and R^(6b) are identical to or different from each other,and denote a hydrogen atom, a substituted or unsubstituted C₁₋₆ alkylgroup, or a substituted or unsubstituted C₆₋₁₄ aryl group), or a groupof the formula —Z¹—Z²

[0061] (wherein Z¹ denotes a single bond, oxygen atom, vinylene group,or ethynylene group;

[0062] Z² denotes a single bond, or a C₁₋₆ alkyl group substituted orunsubstituted with 0 to 4 substituents). R^(3a) and R^(4a) may togetherstand for a methylenedioxy group or 1,2-ethylenedioxy group,alternatively, R^(3a) and R^(4a) may together stand for the formation ofa condensed ring selected from a group consisting of a substituted orunsubstituted benzene ring, substituted or unsubstituted pyridine ring,substituted or unsubstituted pyrrole ring, substituted or unsubstitutedthiophene ring, substituted or unsubstituted furan ring, substituted orunsubstituted pyridazine ring, substituted or unsubstituted pyrimidinering, substituted or unsubstituted pyrazine ring, substituted orunsubstituted imidazole ring, substituted or unsubstituted oxazole ring,substituted or unsubstituted thiazole ring, substituted or unsubstitutedpyrazole ring, substituted or unsubstituted isoxazole ring, substitutedor unsubstituted isothiazole ring, substituted or unsubstitutedcyclohexane ring, and substituted or unsubstituted cyclopentane ring,except in cases where both R^(1a) and R^(2a) stand for hydrogen atoms.]

[0063] 16. The aforementioned antifungal agent of 13, comprising as theactive ingredient compound (Ia) of the formula:

[0064] 17. A compound represented by the general formula (II), a salt ora hydrate thereof, wherein in formula (II),

[0065] [Ar stands for a substituent selected from a group consisting ofthe formulae (IIIa) to (IIIf):

[0066] (wherein K denotes a sulfur atom, oxygen atom, or a grouprepresented by the formula —NH—;

[0067] R^(1b) and R^(2b) are identical to or different from each otherand denote individually a hydrogen atom, halogen atom, hydroxyl group,nitro group, cyano group, trifluoromethyl group, trifluoromethoxy group,a group represented by the formula

[0068] (wherein X³ denotes a single bond, carbonyl group, or a grouprepresented by the formula —S(O)₂—;

[0069] R^(5c) and R^(6c) are identical to or different from each otherand denote a hydrogen atom, or a substituted or unsubstituted C₁₋₆ alkylgroup), or a group represented by the formula —X⁴—R^(8a) (wherein X⁴denotes a single bond, oxygen atom, or sulfur atom; R^(8a) denotes aC₁₋₆ alkyl group, C₂₋₆ alkenyl group, C₂₋₆ alkynyl group, C₃₋₈cycloalkyl group, or C₃₋₈ cycloalkenyl group) Alternatively, R^(1b) andR^(2b) may together form a methylenedioxy group, or a 1,2-ethylenedioxygroup.);

[0070] R^(3b) and R^(4b) are identical to or different from each otherand denote individually a hydrogen atom, halogen atom, hydroxyl group,nitro group, cyano group, carboxyl group, formyl group, hydroxyiminogroup, trifluoromethyl group, trifluoromethoxy group, C₁₋₆ alkyl group,C₁₋₆ alkoxy group, C₂₋₆ alkenyl group, C₂₋₆ alkynyl group, or a grouprepresented by the formula —Z^(1b)—Z^(2b)

[0071] (wherein Z^(1b) denotes a single bond, vinylene group, orethynylene group;

[0072] Z^(2b) denotes a single bond, or a C₁₋₆ alkyl group that issubstituted or unsubstituted with 0 to 4 substituents); except in caseswhere (1) Ar stands for the aforementioned formula (IIId) wherein R^(1b)and R^(2b) are both hydrogen atoms, (2) at least one of R^(3b) or R^(4b)denotes a hydrogen atom and the other is a hydrogen atom, methoxy group,hydroxyl group, methyl group, benzyloxy group, or a halogen atom, and Arstands for the aforementioned formula (IIIc) wherein R^(1b) and R^(2b)both denote hydrogen atoms or methoxy groups, (3) at least one of R^(3b)or R^(4b) denotes a hydrogen atom and the other is a hydrogen atom,hydroxyl group, methoxy group, or benzyloxy group, and Ar stands for theaforementioned formula (IIIc) wherein R^(1b) and R^(2b) both denotehydroxyl groups or benzyloxy groups, or (4) Ar stands for theaforementioned formula (IIId) wherein R^(1b) is a hydrogen atom andR^(2b) is a formyl group, hydroxymethyl group, or methoxycarbonylgroup.]

[0073] 18. The compound of 17, or a salt or hydrate thereof, wherein Arstands for the formula:

[0074] (wherein R^(1c) denotes a hydrogen atom, a substituted orunsubstituted C₁₋₆ alkyl group, or a benzyl group), and excluding thecase when R^(3b) denotes a hydrogen atom.

[0075] 19. A compound represented by the general formula (IIIc2), or asalt or hydrate thereof, wherein in formula (IIIc2),

[0076] [R^(1b) and R^(2b) have the same meaning as defined above, exceptin cases wherein (1) R^(1b) denotes a group represented by the formulaR^(1c)—O— (wherein R^(1c) has the same meaning as defined above), R^(2b)is a hydrogen atom, and R^(3b) denotes a hydrogen atom, (2) at least oneof R^(3b) or R^(4b) denotes a hydrogen atom, and the other is a hydrogenatom, methoxy group, hydroxyl group, methyl group, benzyloxy group, or ahalogen atom, and R^(1b) and R^(2b) both denote hydrogen atoms ormethoxy groups, or (3) at least one of R^(3b) or R^(4b) denotes ahydrogen atom, and the other is a hydrogen atom, hydroxyl group, methoxygroup, or benzyloxy group, and R^(1b) and R^(2b) both denote hydroxylgroups or benzyloxy groups]

[0077] 20. The antifungal agent of 17, having an antifungal action.

[0078] 21. The antifungal agent of 15, wherein at least one of R^(3a)and R^(4a) denotes a group represented by the formula —C(O)NR^(7a)R^(7b)(wherein R^(7a) and R^(7b) have the same meaning as defined above), theformula —CO₂R^(7a) (wherein R^(7a) has the same meaning as definedabove), the formula —S(O)_(n)R^(7a) (wherein n denotes an integer of 0to 2 and R^(7a) has the same meaning as defined above.), the formula—S(O)₂NR^(7a)R^(7b) (wherein R^(7a) and R^(7b) have the same meaning asdefined above), the formula

[0079] (wherein X², R^(5b), and R^(6b) have the same meaning as definedabove), or a C₁₋₆ alkoxy group substituted or unsubstituted with 0 to 4substituents, or R^(3a) and R^(4a) together denote a methylenedioxygroup, or a 1,2-ethylenedioxy group.

[0080] 22. The aforementioned antifungal agent of 15, wherein thecompound having an antifungal action is (1) 1-benzylisoquinoline, (2)1-(4-bromobenzyl)isoquinoline, (3) 1-(4-chlorobenzyl)isoquinoline, (4)1-(4-fluorobenzyl)isoquinoline, (5) 1-(4-iodobenzyl)isoquinoline, (6)1-(3-methylbenzyl)isoquinoline, (7) 1-(4-methylbenzyl)isoquinoline, (8)1-(3,4-dimethylbenzyl)isoquinoline, (9) 1-(3-methoxybenzyl)isoquinoline,(10) 1-(4-methoxybenzyl)isoquinoline, (11)1-(3,4-methylenedioxybenzyl)isoquinoline, (12)1-(4-benzyloxybenzyl)isoquinoline, (13) 1-(4-cyanobenzyl)isoquinoline,(14) 1-(4-nitrobenzyl)isoquinoline, (15) 1-(4-aminobenzyl)isoquinoline,(16) 1-(4-methoxybenzyl)-6,7-dichloro-isoquinoline, (17)1-(4-methoxy-2-nitro-benzyl)-isoquinoline, (18)1-(4-methoxybenzyl)-6,7-methylenedioxy-isoquinoline, (19)1-(2-amino-4-methoxy-benzyl)isoquinoline, (20)1-(4-methoxybenzyl)-7-hydroxy-6-methoxy-isoquinoline, (21)1-(4-benzyloxybenzyl)-6,7-dimethoxy-isoquinoline, (22)1-(4-methoxybenzyl)6,7-dimethoxy-isoquinoline, (23)1(4-methoxy-2-nitro-benzyl)-isoquinoline, (24)3-[4-(1-isoquinolylmethyl)phenoxy]propylcyanide, (25)1-[4-(2,2,3,3-tetrafluoropropoxy)benzyl]isoquinoline, (26)1-[4-(2-piperidinoethoxy)benzyl]isoquinoline, (27)4-(1-isoquinolylmethyl)phenyl(2-morpholinoethyl)ether, (28)1-[4-(2-methoxyethoxy)benzyl]isoquinoline, (29)N-{2-[4-(1-isoquinolylmethyl)phenoxy]ethyl}-N,N-dimethylamine, (30)1-[4-(phenethyloxy)benzyl]isoquinoline, (31)1-{4-[(2-methylallyl)oxy]benzyl}isoquinoline, (32)1-(4-isobutoxybenzyl)isoquinoline, (33)1-[4-(2-phenoxyethoxy)benzyl]isoquinoline, (34) methyl2-[4-(1-isoquinolylmethyl)phenoxy]acetate, (35)2-[4-(1-isoquinolylmethyl)phenoxy]-1-ethanol, (36) t-butylN-{2-[4-(1-isoquinolylmethyl)phenoxy]ethyl}carbamate, (37)1-{4-[3-(tetrahydro-2H-2-pyranyloxy)propoxy]benzyl}isoquinoline, (38)2-[4-(1-isoquinolylmethyl)phenoxy]-1-ethaneamine, (39)1-[4-(3-piperidinopropoxy)benzyl]isoquinoline, (40)3-[4-(1-isoquinolylmethyl)phenoxy]-1-propanol, (41)1-[4-(2-ethylbutoxy)benzyl]isoquinoline, (42)4-[4-(1-isoquinolylmethyl)phenoxy]butanoic acid, (43)1-(4-{3-[(4-benzylpiperazino)sulfonyl]propoxy}benzyl)isoquinoline, (44)1-(4-{3-[4-(4-chlorophenyl)piperazino]propoxy}benzyl)isoquinoline, (45)4-(1-isoquinolylmethyl)aniline, (46)N-[4-(1-isoquinolylmethyl)phenyl]butaneamide, (47)N-[4-(1-isoquinolylmethyl)phenyl]propaneamide, (48)N-[4-(1-isoquinolylmethyl)phenyl]-1-ethanesulfonamide, (49)N-[4-(1-isoquinolylmethyl)phenyl]-N-methyl-ethanesulfonamide, (50)N-[4-(1-isoquinolylmethyl)phenyl]-N-methylamine, (51)N-[4-(1-isoquinolylmethyl)phenyl]-N-propylamine, or (52)N-[4-(1-isoquinolylmethyl)phenyl]-N-methyl-N-propylamine.

[0081] 23. A method for treating a mycotic infection comprisingadministering a therapeutically effective dose of any one of theantifungal agents of 13 to 22 to a mammal.

[0082] The present invention will be described in detail below byexplaining the meaning of the terms, symbols, and such mentioned in thepresent description.

[0083] In the present description, the structural formula of thecompounds may represent a certain isomer for convenience, however, thepresent invention includes all geometrical isomers, optical isomersbased on asymmetric carbon, stereoisomers, and tautomers thatstructurally arise from compounds, and mixtures of isomers, and it isnot to be construed as being limited to the representation in theformula made for convenience, and may be any one or a mixture ofisomers. Therefore, an optically active substance and a racemicsubstance having an asymmetric carbon atom in the molecule may exist,but in this invention there are no particular limitations and any one ofthem are included. Furthermore, crystal polymorphism may exist, butsimilarly there are no limitations, and the crystal form may be any oneform or may be a mixture, and may be either an anhydride or a hydrate.

[0084] Furthermore, the compounds of the present invention includecompounds exhibiting antifungal action after being metabolized, such asafter being oxidized, reduced, hydrolyzed, or conjugated in vivo.Furthermore, the present invention includes compounds that produce thecompounds of this invention after being metabolized, such as after beingoxidized, reduced, and hydrolyzed in vivo.

[0085] The “C₁₋₆ alkyl group” in the present description means astraight chain or branched chain alkyl group, wherein the number ofcarbon ranges from 1 to 6, and specific examples include a methyl group,ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butylgroup, tert-butyl group, n-pentyl group, i-pentyl group, neopentylgroup, n-hexyl group, 1-methylpropyl group, 1,2-dimethylpropyl group,2-ethylpropyl group, 1-methyl-2-ethylpropyl group,1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1-methylbutylgroup, 2-methylbutyl group, 1,1-dimethylbutyl group, 2,2-dimethylbutylgroup, 2-ethylbutyl group, 1,3-dimethylbutyl group, 2-methylpentylgroup, 3-methylpentyl group, and so on.

[0086] The “C₂₋₆ alkenyl group” in the present description means astraight chain or branched chain alkenyl group, wherein the number ofcarbon ranges from 2 to 6, and specific examples include a vinyl group,allyl group, 1-propenyl group, isopropenyl group, 1-butene-1-yl group,1-butene-2-yl group, 1-butene-3-yl group, 2-butene-1-yl group,2-butene-2-yl group, and so on.

[0087] The “C₂₋₆ alkynyl group” in the present description means astraight chain or branched chain alkynyl group, wherein the number ofcarbon ranges from 2 to 6, and specific examples include an ethynylgroup, 1-propynyl group, 2-propynyl group, butynyl group, pentynylgroup, hexynyl group, and so on.

[0088] The “C₁₋₆ alkoxy group” in the present description means an oxygroup to which “C₁₋₆ alkyl group” defined above is bound, and specificexamples include a methoxy group, ethoxy group, n-propoxy group,i-propoxy group, n-butoxy group, i-butoxy group, sec-butoxy group,t-butoxy group, n-pentyloxy group, i-pentyloxy group, sec-pentyloxygroup, t-pentyloxy group, neopentyloxy group, 1-methylbutoxy group,2-methylbutoxy group, 1,1-dimethylpropoxy group, 1,2-dimethylpropoxygroup, n-hexyloxy group, i-hexyloxy group, 1-methylpentyloxy group,2-methylpentyloxy group, 3-methylpentyloxy group, 1,1-dimethylbutoxygroup, 1,2-dimethylbutoxy group, 2,2-dimethylbutoxy group,1,3-dimethylbutoxy group, 2,3-dimethylbutoxy group, 3,3-dimethylbutoxygroup, 1-ethylbutoxy group, 2-ethylbutoxy group, 1,1,2-trimethylpropoxygroup, 1,2,2-trimethylpropoxy group, 1-ethyl-1-methylpropoxy group,1-ethyl-2-methylpropoxy group, and so on.

[0089] The “C₆₋₁₄ aryl group” in the present description refers to anaromatic ring group, wherein the number of carbon ranges from 6 to 14,and specific examples include a phenyl group, 1-naphthyl group,2-naphthyl group, as-indacenyl group, s-indacenyl group, acenaphthylenylgroup, and so on.

[0090] The “halogen atom” of the present description means a fluorineatom, chlorine atom, bromine atom, and iodine atom.

[0091] “Substituted or unsubstituted” in the present description means“the substitutable site may have an arbitrary combination of one or moresubstituents” and specifically the substituents are, for example, ahydrogen atom, halogen, nitro group, cyano group, hydroxyl group,mercapto group, hydroxyalkyl group, carboxyl group, C₁₋₆ alkoxycarbonylgroup, C₂-₇ acylamino group, C₁₋₆ alkylamino group, pyridyl group, C₁₋₆alkylsulfinyl group, C₁₋₆ alkylsulfonyl group, C₁₋₆ alkylsulfamoylgroup, C₁₋₆ alkylsulfinamoyl group, C₁-6 alkylsulfenamoyl group,tetrahydropyranyl group, C₁₋₆ alkylcarbamoyl group, or the formula—X⁴—R^(8a) (wherein X⁴ denotes a single bond, oxygen atom, or sulfuratom; R^(8a) denotes a C₁₋₆ alkyl group, C₂₋₆ alkenyl group, C₂₋₆alkynyl group, C₆₋₁₄ aryl group, C₃₋₈ cycloalkyl group, or C₃₋₈cycloalkenyl group), and so on.

[0092] “May be substituted with 0 to 4 substituents” has the samemeaning as “the substitutable site may have an arbitrary combination of1 to 4 substituents” and the substituents have the same meaning asdefined above.

[0093] “Salt” in the present invention refers to a pharmaceuticallyacceptable salt, and there are no particular limitations as long as thesalt has formed an addition salt with a compound of this invention, anda preferred example is a haloid acid salt such as hydrofluoride,hydrochloride, hydrobromide, and hydroiodide; an inorganic acid saltsuch as a sulfate, nitrate, perchlorate, phosphate, carbonate, andbicarbonate, an organic carboxylate such as an acetate, oxalate,maleate, tartrate, and fumarate; an organic sulfonate such as amethanesulfonate, trifluoromethanesulfonate, ethanesulfonate,benzenesulfonate, toluenesulfonate, and camphorsulfonate; an amino acidsalt such as an aspartate, and glutamate; salts with an amine such as atrimethylamin, triethylamine, procaine, pyridine, andphenethylbenzylamine; alkali metal salts such as sodium, and potassium;alkaline earth metal salts such as magnesium and calcium; and so on.

[0094] Herein below, the following will be disclosed: 1. A method forobtaining DNAs encoding proteins participating in cell wall synthesis,2. a method for examining whether or not a test sample influences theprocess that transports GPI-anchored proteins to the cell wall, and 3. amethod for obtaining the aforementioned compound (Ia) of the presentinvention.

1. A method for Obtaining DNAs Encoding Proteins Participating in FungalCell Wall Synthesis

[0095] Hereinafter, (1) a method for obtaining a DNA encoding a proteinfor acquiring resistance to the aforementioned compound (Ia) byoverexpression in fungi; (2) a method for obtaining a DNA thathybridizes under stringent conditions with the DNA of SEQ ID NO: 1, SEQID NO: 3, or SEQ ID NO: 5; (3) a method for obtaining a DNA that encodesa protein that participates in fungal cell wall synthesis, based on ahomology search; and (4) a method for obtaining a fungus thatoverexpressed or lacked the protein for acquiring resistance to theaforementioned compound (Ia), will be described.

[0096] (1). A Method for Obtaining a DNA Encoding a Protein forAcquiring Resistance to the Aforementioned Compound (Ia) byOverexpression of the DNA in a Fungus

[0097] Herein, “fungus” means a fungus belonging to Division Zygomycota,Ascomycota, Basidiomycota, and Deuteromycota. Preferable is a pathogenicfungus, Mucor, Saccharomyces, Candida, Cryptococcus, Trichosporon,Malassezia, Aspergillus, Trichophyton, Microsporum, Sporothrix,Blastmyces, Coccidioides, Paracoccidioides, Penicillinium, or Fusarium,and more preferable is C. albicans, C. glabrata, C. neoformans, or A.fumigatus. S. cerevisiae and S. pombe, for which genetic analyses areeasy, are also preferred strains.

[0098] A plasmid library of a fungal gene is introduced into a fungus.The plasmid library of S. cerevisiae and S. pombe can be obtained fromATCC (Information for ATCC Number: 37323), and the plasmid library of C.albicans can be produced by the method according to Navaro-Garcia, F. etal, Mol. Cell. Biol., 15: 2197-2206, 1995. The obtained plasmid libraryis introduced to the fungi by the method according to Gietz, D. et al,Nucl. Acids Res. 20: 1425, 1992. Alternatively, a kit such asYEASTMAKER™ Yeast Transformation System (Clontech) may be used.

[0099] The Fungus to which the plasmid library is introduced is culturedin the presence of the aforementioned compound (Ia) Specifically, anagar medium containing the aforementioned compound (Ia) at aconcentration of 1.56 to 25 μg/ml, preferably 1.56 to 6.25 μg/ml, andmore preferably 3.125 μg/ml, is inoculated with the fungus into which aplasmid library has been introduced, is cultured for an appropriatelength of time, at 30° C. to 42° C. for 2 to 5 days, or preferably at37° C. for 3 days. The colony formed upon proliferation is furthercultured in a medium containing the aforementioned compound (Ia), andthe plasmid is purified from the proliferated fungal cells. Purificationof the plasmid can be performed by the method according to METHODS INENZYMOLOGY, Vol. 194: 169-182 (1991), for example.

[0100] Preferably, the nucleotide sequence of the obtained plasmid isdetermined directly, but if necessary, cloning into an appropriatevector, for example pBluescript II, and pUC19 suitable for nucleotidesequence determination, is done to determine the nucleotide sequence. Anucleotide sequence can be determined for example by the methodaccompanying the ABI377 System (PE applied Biosystems) manual.

[0101] In the Examples of the present invention, all 27 of theindependently obtained colonies of S. cerevisiae, and 28 colonies out of30 colonies of C. albicans contained the DNAs of this invention. Onlyone gene that confers resistance to the aforementioned compound (Ia)exists in these fungi and this can be obtained by the abovementionedmethod.

[0102] (2). A Method for Obtaining a DNA That Hybridizes Under StringentConditions to the DNA of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5

[0103] An example of a method for obtaining a DNA encoding a proteinparticipating in fungal cell wall synthesis according to the presentinvention comprises designing a primer from the information of thenucleotide sequence of SEQ ID NO: 1 using the genomic DNA of S.cerevisiae as a template, or designing a primer from the information ofthe nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5 using thegenomic DNA of C. albicans as the template, then performing PCR, andcloning the amplified DNA into an appropriate vector, such aspBlueScript. The primer is designed as necessary according to the regionto be amplified, and the length is preferably 15 bp or more, morepreferably 20 bp or more, and in some cases sequences necessary forsubsequent DNA construction, such as restriction enzyme sites, may beadded. The conditions for PCR can be determined appropriately accordingto factors such as the length of primer, the length of the region to beamplified, and the amount of template DNA to be used. For example, a DNAencoding a protein participating in cell wall synthesis in a fungus canbe obtained using 200 ng of the genomic DNA of C. albicans as atemplate, and SEQ ID NO: 21 and SEQ ID NO: 22 as primers underconditions of 94° C. for 4 minutes→(94° C. for 30 seconds→68° C. for 5minutes)×35 cycles→72° C. for 4 minutes.

[0104] The DNA obtained by PCR may be used as a probe for obtainingother types of fungal DNA showing homology to the DNA encoding theprotein participating in cell wall synthesis. Specifically, for example,to obtain a homologous gene of C. albicans encoding the proteinparticipating in S. cerevisiae cell wall synthesis, DNA that hybridizesunder stringent conditions can be cloned from the genomic library orcDNA library of C. albicans, using the genomic DNA of S. cerevisiae as atemplate, and using DNA that is obtained by PCR as a probe. Herein,stringent conditions refer to hybridization in 4×SSC at 65° C., thenwashing in 0.1×SSC at 65° C. for 1 hour, for example. Furthermore, inanother the stringent conditions are 4×SSC at 42° C. in 50% formamide.Alternatively, conditions such as hybridization in the PerfectHyb™(TOYOBO) solution at 65° C. for 2.5 hours, then washing in 1). 2×SSC,0.05% SDS solution at 25° C. for 5 minutes, 2). 2×SSC, 0.05% SDSsolution at 25° C. for 15 minutes, and 3). 0.1×SSC, 0.1% SDS solution at50° C. for 20 minutes, are also allowed.

[0105] The Examples of this invention demonstrate from Southern Blotanalysis that there is only one gene in C. albicans that hybridizes withthe DNA of SEQ ID NO: 1, and shows the cloning of this gene. From theabove-mentioned method, DNA that hybridizes with SEQ ID NO: 1 or SEQ IDNO: 3 can be obtained.

[0106] (3). A Method for Obtaining a DNA That Encodes a Protein ThatParticipates in Fungal Cell Wall Synthesis, Based on a Homology Search

[0107] The present invention revealed the GWT1 homologues of S.cerevisiae, C. albicans, S. pombe, A. fumigatus, and C. neoformans. Theregion conserved among these genes is considered to be important forGWT1 gene products to exhibit their function, and may very well beconserved in other fungi.

[0108] Therefore, a DNA encoding a protein participating in fungal cellwall synthesis can be obtained by either carrying out hybridization uponconstructing a probe based on the amino acid sequence of the conservedregion, or by performing PCR by designing primers based on the sequence.The PCR primer may be of any sequence as long as it is designed toencode the conserved region, but is preferably SEQ ID NOS: 29 and 31 orpreferably SEQ ID NOS: 29 and 30.

[0109] Furthermore, as another method, a DNA encoding a proteinparticipating in fungal cell wall synthesis can be obtained by carryingout PCR with cDNA or genomic DNA upon finding a nucleotide sequenceshowing homology to GWT1 from gene fragments registered in databases,and then designing primers based on that nucleotide sequence.

[0110] Examples of PCR methods for obtaining a full-length gene based onthe obtained sequence are techniques such as 3′-RACE, 5′-RACE, andinverse PCR, and it is also possible to select by hybridization a clonecontaining neighboring sequences. A full-length gene can be obtained bycombining these techniques.

[0111] (4). A Method for Obtaining a Fungus That Overexpresses or Lacksa Protein for Acquiring Resistance to the Aforementioned Compound (Ia)

[0112] A Fungus, preferably S. cerevisiae, that overexpresses a proteinfor acquiring resistance to the aforementioned compound (Ia) of thisinvention can be obtained by the method of inserting an expressionvector expressing the protein into a particular position on the fungalchromosome, for example an expression vector in which the DNA of SEQ IDNO: 1 is connected downstream of a promoter, which can forcibly expressthe protein in fungi, preferably the promoter of budding yeast enolasegene (ENO1). The insertion method can be performed, for example, by thesteps of, inserting a desired sequence into the multicloning site ofpRS304 (Sikorski R S et al, Genetics. 122(1): 19-27, 1989), constructinga vector for integration, and introducing the vector into the fungus.One can refer to METHODS IN ENZYMOLOGY Vol.194: 281-301 (1991) fordetails.

[0113] Furthermore, an overexpressed strain of C. albicans can beobtained by incorporating the gene of SEQ ID NO: 3 or SEQ ID NO: 5 intoan expression vector for C. albicans, such as pCARS1 and pRM1 (Pla J etal, Yeast 12: 1677-1702, 1996), and then transforming C. albicans(Sanglard D et al, Antimicrobiol. Agents Chemother. 40: 2300-2305,1996).

[0114] Fungi of this invention lacking a gene for acquiring resistanceagainst the aforementioned compound (Ia), preferably S. cerevisiae, canbe obtained by the following methods, but is not to be construed asbeing limited thereto.

[0115] PCR amplification is carried out using a marker gene, preferablyhis5 gene of S. pombe, as a template, and using primers that aredesigned so that PCR products that contain the gene to be deleted (30 bpor more, or preferably 40 bp or more). In the case of S. cerevisiae, thegenetic sequence of SEQ ID NO: 1, positioned on both ends can beobtained. The PCR products can be purified and introduced into fungi,then cultured in a selection medium corresponding to the marker gene,for example, his⁻ for his5, to obtain the deletion strain.

[0116] Furthermore, the deletion strain of C. albicans is obtained bythe usual method using a hisG-URA3-hisG cassette (Fonzi W A et al,Genetics 134: 717-728,1993) based on the nucleotide sequence informationof SEQ ID NO: 3 or SEQ ID NO: 5.

2. A Method for Examining Whether or not the Test Sample Influences theProcess That Transports GPI-Anchored Proteins to the Cell Wall

[0117] Whether or not the test sample inhibits the process thattransports GPI-anchored proteins to the cell wall, or whether or not thetest sample inhibits the expression of the GPI-anchored protein in thefungal surface can be examined by (1) a method using a reporter enzyme,(2) a method using an antibody that reacts with the surface glycoproteinof the fungal cell wall, (3) a method for examining the adhesion abilitytowards animal cells, and (4) a method for observing fungi using anoptical microscope or an electron microscope.

[0118] By using the methods of (1) to (4) described below, preferablythe methods of (1) to (4) in combination, the test sample is judged toinhibit the process that transports GPI-anchored proteins to the cellwall, or the expression of the GPI-anchored proteins at the fungalsurface. Furthermore, it is judged that the test sample influences theprocess that transports GPI-anchored proteins to the cell wall when thedegree of inhibition diminishes or the inhibition is no longer seen whenthe protein encoded by the DNA of the present invention is overexpressedin fungi.

[0119] Hereinafter, the methods of (1) to (4) will be described.

[0120] (1). A Method Using a Reporter Enzyme

[0121] The process that transports GPI-anchored proteins to the cellwall can be quantified by a tracer experiment such as labeling aGPI-anchored protein with a radioactive isotope, then upon fractionationof the fungal cell wall fraction, immunoprecipitating with an antibodyagainst a GPI-anchored protein. Alternatively, the quantification can bemore readily done by expressing the C-terminal sequence considered tofunction as a transport signal, which is commonly observed amongGPI-anchored proteins, as a fusion protein with an easily measurableenzyme (reporter enzyme), fractionating the fungal cell wall fraction,and then using a reporter system that measures the enzyme activity ofeach fraction (Van Berkel M A A et al, FEBS Letters, 349: 135-138,1994). Hereinafter, a method using the reporter enzyme will beexplained, but the present invention is not to be construed as beinglimited thereto.

[0122] First, the reporter gene is constructed and is introduced into afungus. The reporter gene is constructed by linking a promoter sequencethat functions in fungi, followed by DNAs that respectively encode asignal sequence, a reporter enzyme, and a GPI-anchored proteinC-terminal sequence so that the reading frames match. Examples of thepromoter sequences are those of promoters such as GAL10, and ENO1.Examples of signal sequences are those of α-factor, invertase, lysozyme,and such. Examples of reporter enzymes are β-lactamase, lysozyme,alkaline phosphatase, β-galactosidase, and such. Green FluorescenceProtein (GFP), which can be detected easily, can be used, even though itdoes not have enzyme activity. Examples of GPI-anchored proteinC-terminal sequences are α-agglutinin C-terminal sequence, CWP2C-terminal sequence, and such. Furthermore, it is preferable to insertan appropriate selection marker such as LEU2, and URA3 into the vectorcontaining the constructed reporter gene.

[0123] The constructed reporter gene is inserted into a fungus by anappropriate method, such as the lithium acetate method (Gietz D et al,Nucl. Acids Res. 20: 1425, 1992), and cultured, if necessary by a methodsuitable for the selection marker, such as Leu⁻ medium for LEU2, andUra⁻ medium for URA3, and then fungi into which the DNA has beenintroduced are selected.

[0124] Whether or not a test sample influences the process thattransports GPI-anchored proteins to the cell wall is examined by thefollowing method.

[0125] The reporter gene-introduced fungi are cultured under appropriateconditions, for example at 30° C. for 48 hours, in the presence of atest sample. After culturing, the culture supernatant is centrifuged,and the reporter enzyme activity of the culture supernatant fraction ismeasured. The remaining cell fraction is washed, then the cell wallcomponents are separated by an appropriate method, such as degrading thecell wall glucan with glucanase, and then measuring the reporter enzymeactivity of the cell wall fraction and the cytoplasmic fraction. Theassay can be simply carried out by determining the amount of reporterenzyme in the cell fraction by centrifuging, then without washing thecells, determining the amount of reporter enzyme derived from theculture supernatant fraction that remains in the cell fraction byproportional calculation, and subtracting this from the amount ofreporter enzyme of the cell fraction.

[0126] If an activity to increase the reporter enzyme activity withinthe culture supernatant fraction (activity per cell), or an activity todecrease the reporter enzyme activity in the cell wall fraction(activity per cell) is confirmed in the test sample, the test sample isjudged to have influenced the process that transports GPI-anchoredproteins to the cell wall.

[0127] (2). A Method Using an Antibody That Reacts With the SurfaceGlycoprotein of a Fungal Cell Wall

[0128] Whether or not the test sample influences the expression of theGPI-anchored protein at the fungal surface layer can be detected byquantifying a GPI-anchored protein in the fungal cell wall using anantibody that reacts with the protein.

[0129] For example, as the antibody, the antigenic determinant ispredicted from the amino acid sequence of a GPI-anchored protein, forexample, α-agglutinin, Cwp2p, and Als1p (Chen M H et al, J. Biol. Chem.,270:26168-26177, 1995; Van Der Vaat J M et al, J. Bacteriol.,177:3104-3110,1995; Hoyer L L et al, Mol. Microbiol., 15:39-54, 1995),the peptide of that region is synthesized, this is bound to an antigenicsubstance, such as a carrier protein, and then polyclonal antibodies canbe obtained by immunizing a rabbit and such, or a monoclonal antibodycan be obtained by immunizing a mouse and such. Furthermore, a houserabbit polyclonal antibody against the Als1p peptide is preferable.

[0130] In an alternative method, a monoclonal antibody against aGPI-anchored protein may be obtained by immunizing a mouse and such witha fungus, preferably a fungus overexpressing the GPI-anchored protein,such as α-agglutinin, Cwp2p, and Als1p, and in some cases, by immunizingwith the partially purified GPI-anchored protein , and selecting theclone yielded as a result of the fusion by ELISA, Western blot analysis,and such.

[0131] Whether or not the test sample influences the process thattransports GPI-anchored proteins to the cell wall, and diminishes theamount of the protein derived from the GPI-anchored protein in the cellwall can be examined by the following method.

[0132] A fungus is cultured in the presence of a test sample underappropriate conditions, such as 30° C., for 48 hours. The culturedfungus is collected by centrifugation and the cells are disrupted,preferably using glass beads. The washed, disrupted cells are preferablysubjected to centrifugal extraction with SDS, then the precipitate iswashed. After the extraction, the disrupted cells are treated with anenzyme that degrades glucan, preferably glucanase, and the centrifugedsupernatant thereof is the GPI-anchored protein sample.

[0133] The anti-Als1p peptide antibody is coated onto a 96-well plate byincubating at 4° C. overnight. After washing with a washing solution,preferably PBS containing 0.05% Tween 20 (PBST), blocking is carried outwith a reagent that blocks the non-specific adsorption sites of the96-well plate, preferably a protein such as BSA, and gelatin, morepreferably BlockAce. After washing again with a washing solution,preferably PBST, in some cases, after adding an appropriately dilutedGPI-anchored protein sample, the reaction is carried out for anappropriate length of time, such as 2 hours at room temperature. Afterwashing with a washing solution, preferably with PBST, an antibodyagainst the enzyme-labeled C. albicans, preferably HRP-labeledanti-Candida antibody, is reacted for an appropriate length of time,such as 2 hours at room temperature. The method for labeling may beenzyme labeling or radioactive isotope labeling. After washing with awashing solution, preferably PBST, the amount of Als1p in theGPI-anchored protein sample is calculated by a method appropriate forthe type of label, i.e. for an enzyme label, adding a substratesolution, and then upon stopping the reaction, measuring the absorbanceat 490 nm.

[0134] (3). A Method for Examining the Adhesion Ability Towards AnimalCells

[0135] Whether or not the test sample influences expression of aGPI-anchored protein on the fungal surface can be examined by measuringthe activity of the GPI-anchored protein in the fungal cell wall,preferably by measuring the adhesion ability of fungi to animal cells,and such. Besides Als1p, Hwp1p, and such participating in adhesion toanimal cells, α-agglutinin participating in mating, Flo1p participatingin yeast aggregation, and such are known as GPI-anchored proteins.Hereinafter, examination methods that use the adhesion ability of fungito animal cells will be explained in detail, but this invention is notto be construed as being limited thereto.

[0136] As the fungus, a fungus having an adhesion ability towards cellsis used, and preferably, the fungus is C. albicans. For mammalian cells,cells that adhere to the fungus are used, and preferably, are intestinalepithelial cells. The mammalian cells are cultured and are immobilizedby an appropriate method such as ethanol immobilization. The test sampleand the fungi, which have been incubated for an appropriate length oftime, such as 48 hours at 30° C., are inoculated, then after culturingfor a certain length of time, for example 1 hour at 30° C., the culturesupernatant is removed, washed with a buffer, and is superposed onto anagar media, such as Sabouraud Dextrose Agar Medium (Difco). Afterculturing at 30° C. overnight, the number of colonies is counted, andthe adhesion rate is calculated.

[0137] If activity to lower the number of colonies formed by adhesion offungi to cells is observed in a test sample compared to that of fungithat are not treated with the compound, the test sample is judged tohave influenced the process that transports GPI-anchored proteins to thecell wall.

[0138] (4). A Method for Observing Fungi Using an Electron Microscope oran Optical Microscope

[0139] Whether or not a test sample influences the expression of theGPI-anchored proteins in the fungal surface can be examined by observingthe structure of the fungal cell wall using an electron microscope.

[0140] In the presence of a test sample, a fungus such as C. albicans iscultured for a certain length of time, for example, 48 hours at 30° C.,and the ultrafine morphological structure is observed with atransmission electron microscope. Herein, observation with atransmission electron microscope can be carried out, for example by themethod according to the Electron Microscope Chart Manual (MedicalPublishing Center). The flocculent fibrous structure of the outermostlayer of the fungal cell that has a high electron density and isobservable by transmission electron microscope image, is considered tobe a surface glycoprotein layer having GPI-anchored proteins as itsconstituents, and is not influenced by other existing antifungal agents.When this flocculent fibrous structure of the outermost layer of afungal cell, which has a high electron density, disappears leaving aslight layer with a high electron density, compared to that in theuntreated cells, the test sample is judged to have influenced theprocess that transports GPI-anchored proteins to the cell wall.

[0141] When images, in which fungal cells are largely swollen andbudding (division) is inhibited, are observed under a transmissionelectron microscope in addition to an optical microscope, the testsample is judged to have an influence on the cell wall.

[0142] The compounds of the present invention represented by the formula(I)

[0143] (wherein the symbols have the same meaning as defined above) canbe synthesized by utilizing conventional organic chemical reactions andsuch that have been known to date. For example, it can be synthesized bythe following methods.

[0144] In the above formulae, X is a leaving group such as a halogengroup and acyl group. R^(3c) has the same meaning as R^(3a). Othersymbols in the formulae have the same meaning as defined above.

[0145] Process A1

[0146] A reaction for producing the Reissert compound (V). The compoundcan be produced based on the reaction conditions according to theliterature, such as Org. Synth., VI, 115(1988); Heterocycles, 36(11),2489(1993); J. Chem. Soc. (C), 666(1969); or J. Heterocycl. Chem.,29(5), 1165(1992). Specifically, the reagents used are, for example, acombination of benzoyl chloride and potassium cyanide.

[0147] Process A2

[0148] A process for alkylation. The compound (VI) can be produced byreacting the compound (V) with a substituted benzyl halide derivative, asubstituted benzylmethanesulfonate derivative, or such in the presenceof a base. Specific examples of the base include sodium hydride, sodiumhydroxide.

[0149] Process A3

[0150] A process for hydrolysis reaction. The compound (I) can beproduced by hydrolysis of the compound (VI) in the presence of a base.

[0151] Method A is a method for producing the compound (I) via ProcessA1, Process A2, and Process A3.

[0152] Process B1

[0153] A process for conversion of the compound (V) to the compound(VII) The compound (VII) can be produced by reacting the compound (V)with a substituted benzaldehyde in the presence of a base and aphase-transfer catalyst. Examples of the base include sodium hydroxideand potassium hydroxide. Examples of the phase-transfer catalyst includetriethylbenzylammonium chloride.

[0154] Process B2

[0155] A process for oxidation of the alcohol to the ketone. The ketonederivative (VIII) can be produced by using an oxidizing agent and acondition conventionally used for the oxidation reaction of an alcoholto a ketone. Specifically, the oxidizing agent is, for example,manganese dioxide, chromium dioxide, or benzoquinone.

[0156] Process B3

[0157] A process for reduction of the ketone to the methylene. Themethylene derivative (I) can be produced by using a conventionally usedcombination of reducing agents for the reduction reaction of the ketonederivative (VIII) to the methylene derivative (I). Examples of thecombination of the reducing agents include hydrazine hydrate and sodiumhydroxide or potassium hydroxide, triethylsilane and boron trifluoride,and trifluoromethanesulfonic acid.

[0158] Method B is a method for producing the compound (I) via ProcessA1, Process B1, Process B2, and Process B3.

[0159] Process C1

[0160] A process for halogenation or acylation of the hydroxyl group.The compound (IX) can be produced by reacting a halogenating agent or anacylating agent with the compound (VII). Examples of the halogenatingagent include thionyl chloride, concentrated hydrochloric acid, andphosphorus tribromide. Furthermore, examples of the acylating agentinclude acid halides such as acetyl chloride and acid anhydrides such asacetic anhydride.

[0161] Process C2

[0162] A process for reductive elimination reaction of the halogen groupor the acyl group. The compound (I) can be produced by hydroeliminationof the compound (IX), for example, by using a catalyst.

[0163] Examples of the catalyst include palladium-carbon.

[0164] Method C is a method for producing the compound (I) via ProcessA1, Process B1, Process C1, and Process C2.

Production Method (2)

[0165] The compound of the present invention represented by the formula(I) can also be synthesized by the following method.

[0166] In the formula, X is a leaving group such as a halogen group andacyl group. Other symbols in the formulae have the same meaning asdefined above.

[0167] Process D1

[0168] A process for a Grignard reaction and a subsequent acidhydrolysis reaction. The compound (VIII) can be produced by reacting thecompound (X) with a substituted or unsubstituted phenyl Grignardreagent, followed by hydrolysis in the presence of an acid.

[0169] Process D2

[0170] The methylene derivative (I) can be produced from the ketonederivative (VIII) by conditions similar to that of Process B3.

[0171] Method D is a method for producing the compound (I) via ProcessD1 and Process D2.

[0172] Process E1

[0173] A process for the reduction reaction from the ketone to thealcohol. The compound (VII) can be produced from the compound (VIII)using a reducing agent and conditions conventionally used for thereduction reaction of a ketone to an alcohol. Specific examples of thereducing agent include sodium borohydride and lithium aluminum hydride.

[0174] Process E2

[0175] Under conditions similar to that of Process C1, the halogenatedor acylated derivative (IX) can be produced from the alcohol derivative(VII).

[0176] Process E3

[0177] Under conditions for reductive elimination reaction similar tothat of Process C2, the compound (I) can be produced from the compound(IX).

[0178] Method E is a method for producing the compound (I) via ProcessD1, Process E1, Process E2, and Process E3.

Production Method (3)

[0179] The compound of the present invention represented by the formula(I) can also be synthesized by the following method.

[0180] The symbols in the formulae have the same meaning as definedabove.

[0181] Process F1

[0182] A process for the chlorination reaction. The compound (XII) canbe produced by reacting the compound (XI) with a chlorinating agent.Examples of the chlorinating agent include phosphorus oxychloride andthionyl chloride.

[0183] Process F2

[0184] A process for the coupling reaction with a Grignard reagent. Thecompound (I) can be produced by reacting the compound (XII) with asubstituted or unsubstituted benzyl Grignard reagent in the presence ofa catalyst, based on the reaction conditions according to theliterature, such as Arch. Pharm, 314, 156(1981). Examples of thecatalyst include [1,1′-bis(diphenylphosphino)ferrocene]dichloronickel(II).

[0185] Method F is a method for producing the compound (I) via ProcessF1 and Process F2.

Production Method (4)

[0186] The compound of the present invention of the formula (I) whereinR^(1a) and R^(2a) together form a condensed ring such as a benzene ring,pyridine ring, pyrrole ring, thiophene ring, furan ring, cyclohexanering, or cyclopentane ring, can be synthesized by the following method.

[0187] The symbols in the formulae have the same meaning as definedabove.

[0188] The production method in which the isoquinoline ring is formed isshown below as an example.

[0189] Process G1

[0190] A process for the condensation reaction and the subsequentreduction reaction. The compound (XIV) can be produced by a condensationreaction between the substituted or unsubstituted benzaldehydederivative (XIII) and nitromethane, followed by reduction of the nitrogroup. Examples of the reagent used for the reduction of the nitro groupinclude a combination of palladium-carbon and ammonium formate, andlithium aluminum hydride.

[0191] Process G2

[0192] An amide bond formation reaction. The compound (XV) can beproduced by reacting the compound (XIV) and a substituted orunsubstituted phenylacetyl chloride with a coupling reagent for an amidebond formation reaction. Examples of the coupling reagent include acombination of N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide, acombination of N,N′-dicyclohexylcarbodiimide and N-hydroxybenzotriazole,and 1,1′-carbonyldiimidazole.

[0193] Process G3

[0194] A process for the cyclization reaction. The compound (XV) can beproduced based on the reaction conditions according to the literature,such as Organic Reaction, 6, 74(1951); J. Hetetocyclic Chem., 30,1581(1993). Examples of the reagent for this reaction include phosphorusoxychloride and polyphosphoric acid.

[0195] Method G is a method for producing the compound (I) via ProcessG1, Process G2, and Process G3.

Production Method (5-1)

[0196] Replacement of the substituent R^(3a) or R^(4a) of the compound(I) synthesized by the aforementioned production method (5-1)Replacement of the substituent with an amino group, amide group,sulfonamide group, etc.

[0197] The symbols in the formulae have the same meaning as definedabove.

[0198] Process H1

[0199] A reduction reaction of the nitro group. The compound (XVII) canbe produced by reducing the compound (XVI) with a conventionally usedmethod for reduction of a nitro group. Examples of the reduction methodare catalytic hydrogenation reduction by palladium-carbon, or palladiumhydroxide, and reduction by iron-ammonium chloride, iron-hydrochloricacid, iron-acetic acid, etc.

[0200] Process H2

[0201] A process for the acylation or sulfonylation reaction. Thecompound (XVIII) can be produced by treating the compound (XVII) with anacid chloride or acid anhydride.

[0202] Method H is a method for producing the compound (XVIII) viaProcess H1 and Process H2.

[0203] The symbols in the formulae have the same meaning as definedabove.

[0204] Process I1

[0205] A process for the reductive amination reaction. The compound (XX)can be produced from the compound (XIX) and a substituted orunsubstituted aldehyde based on the reaction conditions according to theliterature, such as J. Am. Chem. Soc., 93, 2897(1971); ComprehensiveOrganic Synthese, 8, 25(1991); Tetrahedron, 40, 1783(1984); andTetrahedron, 41, 5307(1985). Examples of the reductive amination reagentinclude sodium triacetoxyhydroborate, sodium cyanotrihydroborate,borane-pyridine complex, and palladium-carbon/hydrogen.

[0206] Process I2

[0207] A process for the acylation, sulfonylation, or reductiveamination reaction. The compound (XXIa) or the compound (XXIb) can beproduced from the compound (XX) using an acid chloride or an acidanhydride. The compound (XXIc) can be produced by carrying out areductive amination reaction similarly to that of Process I1.

[0208] Method I is a method for producing the compound (XXIa), thecompound (XXIb), or the compound (XXIc) via Process I1 and Process I2.

Production Method (5-2)

[0209] Replacement of the Substituent R^(3a) or R^(4a) of the Compound(I) Synthesized by the Aforementioned Production Method

[0210] (5-2) Replacement of the Substituent With a Hydroxyl Group,Alkoxy Group, etc.

[0211] The symbols in the formulae have the same meaning as definedabove.

[0212] Process J1

[0213] The compound (XXIII) can be produced from the compound (XXII) bya demethylation reaction based on the reaction conditions according tothe literature, such as Bull. Chem. Soc. Jpn., 44, 1986(1971); Org.Synth., Collect. Vol. V, 412(1073); J. Am. Chem. Soc., 78, 1380 (1956);or J. Org. Chem., 42, 2761 (1977). Examples of the reagent used for thedemethylation reaction include 47% aqueous hydrobromic acid solution,boron tribromide, pyridine hydrochloride, and iodotrimethylsilane.

[0214] Process J2

[0215] A process for the alkylation reaction. The compound (XXIV) can beproduced by reacting the compound (XXIII) with a substituted orunsubstituted alkyl halide, a substituted or unsubstituted alkylmethanesulfonate, or such in the presence of a base.

[0216] Method J is a method for producing the compound (XXIV) viaProcess J1 and Process J2.

Production Method (5-3)

[0217] Replacement of the Substituent R^(3a) or R^(4a) of the Compound(I) Synthesized by the Aforementioned Production Method

[0218] (5-3) Replacement of the Substituent With a Vinylene Group, anEthynylene Group, Alkyl Group, etc.

[0219] The symbols in the formulae have the same meaning as definedabove.

[0220] Process K1

[0221] A process for the triflation reaction. The compound (XXV) can beproduced by reacting the compound (XXIII) with trifluoromethane sulfonicacid anhydride in the presence of a base.

[0222] Process K2

[0223] A process for the coupling reaction with an alkyne. The compound(XXVI) can be produced by coupling the compound (XXV) with an alkynederivative in the presence of a palladium phosphine complex, copperiodide, and a base. Examples of reagents that produce the palladiumphosphine complex in the reaction system include a combination ofpalladium-carbon and triphenylphosphine, tetrakistriphenylphosphinepalladium (0) and triphenylphosphine, dichlorobistriphenylphosphinepalladium (II), palladium (II) acetate and tri(o-tolyl)phosphine, andpalladium(II) acetate and 1,1′-bis (diphenylphosphino) ferrocene.Examples of the base include triethylamine, piperidine, pyridine, andpotassium carbonate. Depending on the reaction, lithium chloride may beused.

[0224] Process K3

[0225] A process for the reduction reaction of the unsaturatedhydrocarbon. The compound (XXVIIa) or the compound (XXVIIb) can beproduced from the compound (XXVI), for example, by catalytichydrogenation using a catalyst. Examples of the catalyst includepalladium-carbon, palladium hydroxide, platinum oxide, andpalladium-carbon-calcium carbonate.

[0226] The symbols in the formulae have the same meaning as definedabove.

[0227] Process L1

[0228] A process of the coupling reaction (Heck Reaction) with thealkene. The compound (XXVIIa) can be produced from the compound (XXVIII)using a catalyst (e.g. palladium complex and its ligand), based on thereaction conditions according to the literature, such as J. Org. Chem.37, 2320 (1972); Org. Reactions., 27, 345 (1982); Comprehensive OrganicSynthesis, Vol. 4, 833 (1991); Palladium Reagents and Catalysts, 125(1995); Chem. Commun., 1287(1984); Tetrahedron Lett, 26, 2667 (1985);and Tetrahedron Lett, 31, 2463 (1990). Examples of the combination ofthe catalysts used for this reaction (palladium complex and its ligand)include palladium (II) acetate and 1,1′-bis(diphenylphosphino)ferrocene,and palladium (II) acetate and tri(o-tolyl)phosphine. Examples of thetertiary base include triethylamine, diisopropylethylamine, and1,8-diazabicyclo[5.4.0]-7-undecene. X of the compound (XXVIII) denotes aleaving group, such as a halogen group and trifluoromethanesulfonyloxygroup.

[0229] Process L2

[0230] The compound (XXVIIb) can be produced from the compound (XXVIIa)according to the conditions for a reduction reaction of an unsaturatedhydrocarbon, similar to that of process K3.

[0231] Method L is a method for producing the compound (XXVIIa) byProcess L1, followed by producing the compound (XXVIIb) by Process L2.

[0232] Various isomers of the compounds represented by the formula (I)of the present invention can be purified and isolated using ordinaryseparation techniques (for example, recrystallization, chromatography,and so on).

[0233] Compounds of the present invention or salts thereof, or hydratesthereof can be administered as they are to mammals (preferably humans)They can also be formulated by a conventional method into tablets,powders, fine granules, granules, coated tablets, capsules, syrups,troches, inhalants, suppositories, injections, ointments, eye ointments,eye drops, nasal drops, ear drops, cataplasms, lotions, and such, thenadministered. For the pharmaceutical formulation, ordinarily usedauxiliary agents for pharmaceutical formulation (for example, fillers,binders, lubricants, coloring agents, flavoring agents, and asnecessary, stabilizers, emulsifiers, absorbefacient, surfactants, pHregulators, antiseptics, antioxidants, etc.) can be used. Thepharmatical formulation can be prepared by an ordinary method bycombining components that are generally used as ingredients forpharmaceutical preparations. For example, oral preparations can beproduced by combining the compounds of the present invention or apharmaceutically acceptable salt thereof with fillers, and as necessary,binders, disintegrators, lubricants, coloring agents, flavoring agents,and such, and formulating the mixture into powders, fine granules,granules, tablets, coated tablets, capsules, and such by usual methods.Examples of these components include animal fat and vegetable oil suchas soybean oil, beef tallow, and synthetic glyceride; hydrocarbons suchas liquid paraffin, squalene, and solid paraffin; ester oils such asoctyldodecyl myristate and isopropyl myristate; higher alcohols such ascetostearyl alcohol and behenyl alcohol; silicone resin; silicone oil;surfactants such as polyoxyethylene fatty acid ester, sorbitan fattyacid ester, glycerol fatty acid ester, polyoxyethylene sorbitan fattyacid ester, polyoxyethylene hardened castor oil, and polyoxyethylenepolyoxypropylene block copolymer; water-soluble macromolecules such ashydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer,polyethylene glycol, polyvinyl pyrrolidone, and methyl cellulose; loweralcohols such as ethanol and isopropanol; polyhydric alcohols such asglycerol, propylene glycol, dipropylene glycol, and sorbitol; sugarssuch as glucose and sucrose; inorganic powder such as silicic acidanhydride, magnesium aluminum silicate, and aluminum silicate; purifiedwater, etc. Examples of fillers include lactose, corn starch,refinedwhite sugar, glucose, mannitol, sorbitol, crystalline cellulose,and silicon dioxide. Examples of binders include polyvinyl alcohol,polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic,tragacanth, gelatin, shellac, hydroxypropylmethyl cellulose,hydroxypropyl cellulose, polyvinyl pyrrolidone, polypropyleneglycolpolyoxyethylene block polymer, and meglumine. Examples of disintegratorsinclude starch, agar, powdered gelatin, crystalline cellulose, calciumcarbonate, sodium hydrogencarbonate, calcium citrate, dextrin, pectin,and calcium carboxymethylcellulose. Examples of lubricants includemagnesium stearate, talc, polyethyleneglycol, silica, and hardenedvegetable oil. Examples of coloring agents are those accepted foraddition to medicaments. Examples of flavoring agents include cocoapowder, l-menthol, aromatic dispersant, mint oil, borneol, and cinnamonpowder. The use of sugar coating and other appropriate coating asnecessary is of course permissible for these tablets and granules.Furthermore, liquid preparations such as syrups and injections can beprepared using conventional methods by adding pH regulators,solubilizers, isotonizing agents, and such, and as necessary,solubilizing adjuvants, stabilizers, and such to the compounds of thisinvention or pharmaceutically acceptable salts thereof. The method forproducing external preparations is not limited and can be produced by aconventional method. That is, base materials used for formulation can beselected from various materials ordinarily used for medicaments,quasi-drugs, cosmetics, and such. Specifically, the base materials to beused are, for example, animal fat and vegetable oil, mineral oil, esteroil, waxes, higher alcohols, fatty acids, silicone oil, surfactants,phospholipids, alcohols, polyhydric alcohols, water solublemacromolecules, clay minerals, and purified water. As necessary, pHregulators, antioxidants, chelating agents, antiseptic and antifungalagents, coloring matters, fragrances, and such may be added, but thebase materials of the external preparations of the present invention arenot to be construed as being limited thereto. Furthermore, as necessary,components such as those that have a differentiation induction effect,blood flow accelerants, fungicides, antiphlogistic agents, cellactivators, vitamins, amino acids, humectants, and keratolytic agentscan be combined. The above-mentioned base materials is added to anamount that leads to the concentration usually used for externalpreparations.

[0234] When the compounds of this invention or salts thereof, orhydrates thereof, is administered, there are no particular limitationson their form, and they can be administered orally or parenterally by aconventionally used method. They can be formulated into as dosage formssuch as tablets, powder, fine granules, capsules, syrups, troches,inhalents, suppositories, injections, ointments, eye ointments, eyedrops, nasal drops, ear drops, cataplasms, and lotions. The dose of thepharmaceutical compositions of this invention can be selectedappropriately depending on the degree of the symptom, age, sex, weight,the dosage form, the type of salt, the specific type of disease, andsuch.

[0235] A curative dose of the antifungal agent of this invention isadministered to a patient. Herein, “curative dose” refers to the amountof the pharmaceutical agent that yields the desired pharmacologicalresult and is effective for recovery or relief from the symptoms of apatient to be treated. The dose differs markedly depending on the weightof the patient, type of disease, degree of symptom, age of the patient,sex, sensitivity towards the agent, and such. Usually, the daily dosefor an adult is approximately 0.03 to 1000 mg, preferably 0.1 to 500 mg,more preferably 0.1 to 100 mg, and is administered once to several timesper day, or once to several times per several days. The dose forinjections is normally, approximately 1 to 3000 μg/kg, and is preferablyapproximately 3 to 1000 μg/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0236]FIG. 1 is a schematic diagram of the process that transportsGPI-anchored proteins to the cell wall. A GPI(Glycosylphosphatidylinositol)-anchored protein is first anchored toGPI, and then transported to the cell wall.

[0237]FIG. 2 is a graph showing the activity of the aforementionedcompound (Ia) in the S. cerevisiae reporter system. In the presence ofthe aforementioned compound (Ia) at a concentration of 0.39 to 1.56μg/ml, cephalosporinase activity increased in the culture supernatantfraction and decreased in the cell wall fraction, and at a concentrationof 3.13 μg/ml or more, growth inhibition was observed.

[0238]FIG. 3 is a graph showing the effect of the aforementionedcompound (Ia) on the adhesion of C. albicans to animal cells. Even at aconcentration of 1.56 μg/ml in which growth inhibition cannot beobserved, adhesion of C. albicans to animal cells was inhibited to abouta half.

[0239]FIG. 4 is a graph showing the effect of the aforementionedcompound (Ia) on the amount of the Als1p antigen of C. albicans. In thepresence of the aforementioned compound (Ia) at a concentration of 0.1to 0.39 μg/ml, the amount of the Als1p antigen increased in the culturesupernatant fraction and the amount of the antigen decreased in the cellwall fraction.

[0240]FIG. 5 is a photograph showing the Southern Blot analysis of theC. albicans gene using the GWT1 gene as a probe. A single band wasobserved at 6.5 kb with EcoRI, at 4.0 kb with HindIII, at 2.0 kb withEcoRI-HindIII, and at 2.5 kb with EcoRI-PstI, and the homologue of theresistant gene to the aforementioned compound (Ia) in C. albicans wasexpected to exist as a single gene.

[0241]FIG. 6 is a graph showing the activity of the aforementionedcompound (Ia) in S. cerevisiae that overexpressed the GWT1 gene product.In S. cerevisiae CW63 strain (“W/T” in the Figure), even at theconcentration of the aforementioned compound (Ia) (0.39 to 1.56 μg/ml)in which cephalosporinase activity in the culture supernatant fractionis increased, and activity in the cell wall fraction is decreased, suchan effect was not observed in S. cerevisiae CW63/GWT1 strain, and in S.cerevisiae CW63 strain, even at the concentration of the aforementioned(>3.13 μg/ml) in which growth is inhibited, growth inhibition was notobserved in S. cerevisiae CW63/GWT1 strain (“O/E” in the Figure).

[0242]FIG. 7 is a diagram in which the highly conserved regions in theproteins encoded by the GWT1 genes of S. cerevisiae, S. pombe, and C.albicans are aligned.

BEST MODE FOR CARRYING OUT THE INVENTION Example A

[0243] The present invention is specifically illustrated below withreference to Examples, but it is not to be construed as being limitedthereto.

Example A1 Construction of the Reporter Gene and Introduction Thereofinto S. cerevisiae

[0244] (1). Construction of the Reporter Gene Where Lysozyme is theReporter Enzyme

[0245] A lysozyme gene comprising a promoter sequence was amplified byPCR using pESH plasmid comprising the ENO1 promoter + secretion signal +the lysozyme gene (Ichikawa K et al, Biosci. Biotech. Biochem., 57 (10),1686-1690, 1993) as template, and the oligonucleotides of SEQ ID NO: 8and SEQ ID NO: 9 as primers, and this was subcloned into the SalI-EcoRIsite of pCR-Script SK(+) (a). Furthermore, a CWP2 gene was amplified byPCR using S. cerevisiae chromosomal DNA as template, and theoligonucleotides of SEQ ID NO: 10 and SEQ ID NO: 11 as primers, and thiswas subcloned into the EcoRI-HindIII site of pUC19 (b). Similarly, CYC1terminator was amplified by PCR using pYES2 (INVITROGEN) as a template,and the oligonucleotides of SEQ ID NO: 12 and SEQ ID NO: 13 as primers,and this was subcloned into the newly introduced NotI-KpnI site of pUC19(c).

[0246] Next, the lysozyme gene excised with SalI-EcoRI (a), and the CWP2gene excised with EcoRI-HindIII (b) were inserted into the SalI-HindIIIcleavage site of pESH. Finally, pRLW63T was produced by excising a genecomprising the ENO1 promoter + secretion signal + lysozyme gene + CWP2gene using BamHI-HindIII, inserting this into a pRS306 integrationvector (Sikorski R S et al, Genetics. 122(1): 19-27, 1989), and theninserting the CYC1 terminator excised with HindIII-KpnI (c) into theHindIII-KpnI cleavage site.

[0247] (2). Construction of the Reporter Gene Where Cephalosporinase isthe Reporter Enzyme

[0248] DNA comprising a promoter sequence and secretion signal portionwas amplified by PCR using the abovementioned pESH as template, the ENO1promoter C-terminus + secretion signal portion (d) as template, and theoligonucleotides of SEQ ID NO: 14 and SEQ ID NO: 15 as primers, and thiswas subcloned into the BamHI-NotI site newly introduced into pUC19 (d).Furthermore, a cephalosporinase gene was amplified by PCR usingCitrobacter freundii chromosomal DNA as template, and theoligonucleotides of SEQ ID NO: 16 and SEQ ID NO: 17 as primers, and thiswas subcloned into the NspV-XbaI site newly introduced into pUC19 (e).Similarly, the CWP2 gene was amplified by PCR using the S. cerevisiaechromosomal DNA as template, and the oligonucleotides of SEQ ID NO: 18and SEQ ID NO: 19 as primers, and this was subcloned into theXbaI-HindIII site of pUC19 (f).

[0249] After producing the full length ENO1 promoter + secretion signalportion by inserting the BamHI-SalI fragment of pESH into the BamHI-SalIcleavage site of a plasmid into which (d) has been inserted, thecephalosporinase gene excised with NspV-XbaI, and the CWP2 gene excisedwith XbaI-HindIII were inserted into the NspV-HindIII cleavage site.Next, pRCW63T was produced by excising with EcoRI-HindIII, insertingthis fragment into the abovementioned pRS306, and then inserting theCYC1 terminator into the HindIII-KpnI cleavage site.

[0250] (3). Introduction of the Reporter Gene Into S. cerevisiae

[0251]S. cerevisiae G2-10 strain was cultured by shaking in 10 ml of YPDmedium at 30° C., then the cells were collected at the late logarithmicgrowth phase (2-5×10⁷ cells/ml). After washing with sterilized water,the above mentioned pRLW63T and pRCW63T were introduced by lithiumacetate method that uses YEASTMAKER™ Yeast Transformation System(Clontech) (according to the YEASTMAKER™ Yeast, Transformation SystemUser Manual). pRLW63T and pRCW63T in which the URA3 gene was cleavedwith EcoRV and ApaI, respectively, were used. After culturing inSD(Ura⁻) medium at 30° C. for 3 days, the grown colonies were culturedin YPD medium.

[0252] When the localizations of lysozyme and cephalosporinaseactivities were confirmed, both activities were mainly localized in thecell wall, and the C-terminal sequence of CWP2 was confirmed to functionas a transport signal to the cell wall.

Example A2 Screening of Pharmaceutical Agents by the S. cerevisiaeReporter System

[0253] Since sensitivity of the enzyme reaction is better withcephalosporinase compared to lysozyme, S. cerevisiae introduced withpRCW63T (S. cerevisiae CW63 strain) was used for the screening ofcompounds.

[0254] After stationary cultivation in YPD liquid medium at 30° C. for48 hours, the yeast cell culture was diluted 100 times with YPD liquidmedium (3-5×10⁵ cells/ml) and 75 μl/well aliquots thereof wereinoculated into a V-bottomed 96-well plate containing 25 μl/well of adiluted test sample, and this was subjected to stationary cultivation at30° C. for 48 hours. After centrifuging the plate, 25 μl of thesupernatant was sampled and placed in a flat-bottomed 96-well plate, andthis was used as the culture supernatant fraction.

[0255] The precipitated cells were suspended, and 75 μl/well aliquots ofZymolyase (Seikagaku Corporation) solution prepared with 2.4 M sorbitolwere added and were allowed to react at 30° C. for 1 hour. Aftercentrifuging the plate, 10 μl of the supernatant was sampled and placedin a flat-bottomed 96-well plate, 15 μl of phosphate buffer was added,and this was used as the cell wall fraction.

[0256] The cephalosporinase activities in the medium and in the cellwall fraction were measured by adding 200 μM of nitrocefin solution to apooled sample, and after a certain period of time, stopping the reactionwith citric acid buffer, and then measuring the absorbance at 490 nm.

[0257] Furthermore, fungal growth in the presence of the test sample wasdetermined by visual observation.

[0258]FIG. 2 showed that in the presence of the aforementioned compound(Ia) at a concentration of 0.39 to 1.56 μg/ml, cephalosporinase activityincreases in the culture supernatant fraction, and the activitydecreases in the cell wall fraction. In this manner, a compound thatincreases the cephalosporinase activity in the culture supernatantfraction, and in addition decreases the cephalosporinase activity in thecell wall fraction was considered to be a compound that inhibits theprocess that transports GPI-anchored proteins to the cell wall.

Example A3 Screening of Pharmaceutical Agents Using the Adhesion ofCandida to Animal Cells

[0259] Three-milliliter aliquots of IEC-18 cells (1×10⁵ cells/ml inD-MEM medium (Nissui Pharmaceutical) containing 10% fetal calf serum and2 mM glutamine) were placed in each well of a 6-well multi-well plate.The plate was incubated in a carbon dioxide gas incubator at 37° C. for3 days, the culture supernatant was removed, and ethanol immobilizationwas carried out.

[0260]C. albicans cultured in Sabouraud Dextrose Liquid Mediumcontaining various concentrations of the test sample at 30° C. for 48hours was adjusted to 4×10² cells/ml, and 1 ml was inoculated into eachwell of the plate in which the immobilized IEC-18 cells were cultured.After cultivation at 30° C. for 1 hour, the culture supernatant wasremoved, washed with PBS, and then 2 ml of Sabouraud Dextrose AgarMedium (Difco) was superposed. After cultivation at 30° C. overnight,the number of colonies (CFU) that had grown was counted and the adhesionrate was calculated.

[0261]FIG. 3 shows that even at a concentration of 1.56 μg/ml of theaforementioned compound (Ia), in which growth inhibition cannot beobserved, adhesion of C. albicans to animal cells was inhibited to abouta half. Compared to untreated C. albicans, a test sample that diminishedCFU that adhered to cells was considered as a compound that inhibits theadhesion of C. albicans to animal cells.

Example A4 Screening of Pharmaceutical Agents Using the Amount of theGPI-Anchored Protein Quantified by ELISA

[0262] (1). Production of Anti-Als1p Peptide Antibody

[0263] A house rabbit was immunized with the synthetic peptide of SEQ IDNO: 20 which was conjugated with KLH. The obtained antisera wasaffinity-purified, and the IgG fraction was used as the anti-Als1ppeptide antibody.

[0264] (2). Screening of Pharmaceutical Agents by ELISA Using Anti-Als1pPeptide Antibody

[0265]C. albicans was cultured in Sabouraud Dextrose Liquid Medium (5ml) containing various concentrations of the test sample at 30° C. for48 hours, and the cells were collected by centrifugation, washed, andthen suspended in 300 μl of Tris-HCl buffer. The suspended cells weretransferred to a microtube containing glass beads, and were disrupted byrepeating 10 cycles of stirring for 1 minute and cooling on ice for 1minute. The disrupted cells that were washed were extracted with 2% SDSat 95° C. for 10 minutes, centrifuged, and then the precipitate waswashed 5 times with phosphate buffer. To this precipitate, 0.5 ml of 5μg/ml Zymolyase solution was added, reacted at 37° C. for 1 hour, andthe centrifuged supernatant was used as the GPI-anchored protein sample.

[0266] A 96-well plate was coated with 50 μL of anti-Als1p peptideantibody (40 μg/ml) at 4° C. overnight. After washing 5 times with PBScontaining 0.05% Tween 20 (PBST), blocking was carried out with 25%BlockAce at room temperature for 2 hours. After washing 3 times withPBST, 50 μl of the 2-fold serially diluted GPI-anchored protein samplewas reacted at room temperature for 2 hours. After washing 5 times withPBST, 100 μl of 1000-fold diluted HRP-labeled anti-Candida antibody(ViroStat) was reacted at room temperature for 2 hours, then uponwashing 5 times with PBST, 75 μl of substrate solution was added. Afterthe reaction was stopped, absorbance at 490 nm was measured.

[0267]FIG. 4 shows that in the presence of the aforementioned compound(Ia) at a concentration of 0.1 to 0.39 μg/ml, the amount of Als1pantigen increases in the culture supernatant fraction, and the amount ofantigen decreases in the cell wall fraction. In this manner, a compoundthat increased the amount of Als1p in the culture supernatant, ordecreased the amount of Als1p in the cell wall fraction, as quantifiedby ELISA, compared to the amount of Als1p in C. albicans untreated withthe compound, was considered to be a compound that inhibits the processthat transports GPI-anchored proteins to the cell wall in C. albicans.

Example A5 Observation of the Cell Wall of C. albicans Cultured in thePresence of a Test Sample by an Electron Microscope

[0268]C. albicans which was cultured in Sabouraud Dextrose Liquid Medium(5 ml) containing various concentrations of the test agent at 30° C. for48 hours, then centrifuged, and collected, was immobilized by potassiumpermanganate immobilization method, and the transmission electronmicroscope image thereof was observed.

[0269] The flocculent fibrous structure with high electron density wasobserved in the outermost layer of the cell, and was considered to bethe surface layer glycoprotein layer having the GPI-anchored protein asits constituent. This flocculent fibrous structure was not influenced byother existing antifungal agents.

[0270] In C. albicans cultured in the presence of the aforementionedcompound (Ia), the flocculent fibrous structure of the outermost layerof the cell having high electron density disappeared leaving a smallamount of the layer with high electron density, compared to that inuntreated cells. In this manner, when the flocculent fibrous structureof the outermost layer of the fungal cell having high electron densitydisappeared, the test sample was considered to be the compoundinfluencing the process that transports GPI-anchored proteins to thecell wall.

Example A6 Screening of the Resistant Gene to the AforementionedCompound (Ia) of S. cerevisiae

[0271] The plasmid library of the S. cerevisiae gene was obtained fromATCC (Information for ATCC Number: 37323).

[0272]S. cerevisiae G2-10 strain was cultured while shaking in 10 ml ofYPD medium at 30° C., and cells were collected at the late logarithmicgrowth phase (1-2×10⁷ cells/ml). After washing the cells with sterilizedwater, the plasmid library of the S. cerevisiae gene was introduced bythe lithium acetate method that uses YEASTMAKER™ Yeast TransformationSystem (Clontech) (according to YEASTMAKER™ Yeast Transformation SystemUser Manual), and this was spread onto a SD(Leu⁻) plate, andapproximately 80,000 colonies were obtained. The colonies were collectedand diluted, and were spread onto a SD(Leu⁻) plate containing theaforementioned compound (Ia) at a concentration of 1.56 μg/ml and 3:125μg/ml so that there were 570,000 colonies per plate. Subsequently, theresistant clone was obtained by incubation at 37° C. for 72 hours.

[0273] When 27 clones were picked and plasmids were collected by themethod according to METHODS IN ENZYMOLOGY, Vol. 194: 169-182 (1991), andthe inserts were analyzed, all 27 contained the same fragment.

[0274] As a result of determining the nucleotide sequence using theABI377 system (PE Applied Biosystems), the DNA of SEQ ID NO: 1 was foundto be the DNA that confers resistance to the aforementioned compound(Ia), and was named GWT1.

Example A7 Southern Blot Analysis of a C. albicans Homologue of the S.cerevisiae GWT1 Gene

[0275] A sample was prepared by treating 25 μg of the C. albicansgenomic DNA with EcoRI (TaKaRa), HindIII (TaKaRa), BamHI (TOYOBO), orPstI (New England Biolabs) (including a combination of 2 types ofenzymes) for 16 hours, then concentrating by ethanol precipitation, anddissolving in 25 μl of sterilized water. Twenty-five micrograms ofgenomic DNA digested with restriction enzymes was separated by 0.75%agarose gel electrophoresis method, and was transferred to a nylonmembrane (GeneScreen PLUS/NEN).

[0276] A probe was produced by labeling 20 ng of the approximately 1.5kb DNA fragment of SEQ ID NO: 1 with alpha33P-dCTP by the random primermethod, and was purified using a GeneQuant column (Amersham-Pharmacia).

[0277] Hybridization was carried out by soaking the membrane in 10 ml ofPerfectHyb™ (TOYOBO) solution, preincubating at 65° C. for 1 hour, thenadding the labeled probe mentioned above, and incubating at 65° C. for2.5 hours. Washing was carried out with 1). 2×SSC, 0.05% SDS solution at25° C. for 5 minutes, 2). 2×SSC, 0.05% SDS solution at 25° C. for 15minutes, and 3). 0.1×SSC, 0.1% SDS solution at 50° C. for 20 minutes.The washed membrane was wrapped with Saran Wrap, and contacted with anImaging Plate (FUJI) for 12 hours at room temperature, the image thatwas transferred to the Imaging Plate was captured using BAS2000 (FUJI),and the image was analyzed.

[0278] As a result, single bands were observed at 6.5 kb with EcoRI, 4.0kb with HindIII, 2.0 kb with EcoRI-HindIII, and 2.5 kb with EcoRI-PstI(FIG. 5), and the homologue of the resistant gene to the aforementionedcompound (Ia) of C. albicans was expected to exist as a single gene.

Example A8 Screening of the Resistant Gene to the AforementionedCompound (Ia) of C. albicans

[0279] The genomic library of C. albicans was produced by the methodaccording to Navaro-Garcia F et al, Mol. Cell. Biol., 15: 2197-2206,1995. Specifically, the genomic DNA of C. albicans was partiallydigested with Sau3AI, then DNA fragments around 3 to 5 were collected,and these were inserted into the BamHI site of YEp352 shuttle vector.

[0280]S. cerevisiae G2-10 strain was cultured by shaking in 10 ml of YPDmedium at 30° C., and cells were collected at the late logarithmicgrowth phase (2-5×10⁷ cells/ml). After washing the cells with sterilizedwater, a genomic library of the C. albicans was introduced by thelithium acetate method that uses YEASTMAKER™ Yeast Transformation System(Clontech) (according to YEASTMAKER™ Yeast Transformation System UserManual), and this was spread onto a SD(Ura⁻) plate, and approximately25,000 colonies were obtained. The colonies were collected and diluted,and were spread onto a SD plate containing the aforementioned compound(Ia) at a concentration of 1.56 μg/ml so that there were 500,000colonies per plate. Subsequently, the resistant clones were obtained byincubation at 30° C. for 6 hours, and then transferred to 37° C. andincubated for 66 hours.

[0281] When 30 clones were picked and plasmids were collected by themethod according to METHODS IN ENZYMOLOGY, Vol. 194: 169-182 (1991) andthe inserts were analyzed, 28 out of 30 contained the same fragment.

[0282] As a result of determining the nucleotide sequence using theABI377 system (PE Applied Biosystems), the DNA of SEQ ID NO: 3 was foundto be the DNA that confers resistance to the aforementioned compound(Ia).

Example A9 Cloning of a Homologue of the Resistant Gene to theAforementioned Compound (Ia) From the Clinical Isolate of C. albicans

[0283] PCR amplification was carried out using as template a genomic DNAthat was purified from a clinical isolate of C. albicans that is storedby the inventors, and SEQ ID NO: 21 and SEQ ID NO: 22 as primers. A DNAfragment of approximately 1.6 kb was amplified from all three of theindependent PCR samples, the amplified fragments were purified,subcloned into a pT7-Blue vector (Novagen), and the nucleotide sequencewas determined, and thereby, the DNA sequence of SEQ ID NO: 5 wasdiscovered. The sequence was different at three positions as compared tothe DNA of Example A7 (SEQ ID NO: 3).

[0284] Furthermore, in the nucleotide sequence of the C. albicans genedetermined at Stanford University Sequence Center(http://sequence-www.stanford.edu/), a homologue of the DNA of ExampleA7 was found (SEQ ID NO: 7), and the sequence was different at fourpositions as compared to the DNA of Example A7 (SEQ ID NO: 3).

Example A10 Construction of S. cerevisiae Overexpressing the GWT1 GeneProduct

[0285] PCR amplification was carried out using a plasmid purified fromthe resistant clone to the aforementioned compound (Ia) obtained inExample A6 as a template, and SEQ ID NO: 23 and SEQ ID NO: 24 asprimers. A PCR product cleaved with PvuII was inserted into theSalI-HindIII cleavage site of pRLW63T produced in Example A1. The entireinsert was excised with BamHI-KpnI, and was inserted into the MCS(multi-cloning site) of pRS304 (Sikorski R S et al, Genetics. 122(1):19-27, 1989) to produce a vector for integration.

[0286]S. cerevisiae CW63 strain having a cephalosporinase gene as thereporter gene was cultured by the method according to Example A1, TRP1of the integration vector was cleaved with EcoRV, and thentransformation was carried out by the method of Example A1.GWT1-overexpressed strain (S. cerevisiae CW63/GWT1 strain) was obtainedby culturing in SD(Trp⁻) medium at 30° C. for 3 days.

[0287] Other than showing resistance to the aforementioned compound(Ia), GWT1-overexpressed strain is not different from the wild typestrain, and was sensitive towards other antifungal agents,cycloheximide, benomyl, and amphotericin B.

Example A11 Construction of S. cerevisiae Mutant Lacking the GWT1 Gene

[0288] His5 cassette containing the GWT1 sequence on both ends wasamplified by PCR using the his5 gene of S. pombe (Longtine M S et al,Yeast, 14: 953-961, 1998) as template and SEQ ID NO: 25 and SEQ ID NO:26 as primers.

[0289]S. cerevisiae G2-10 was cultured and the cells were collected bythe method according to Example A1, and the abovementioned PCR productwas transformed by the method according to Example A1. A GWT1-deficientstrain was obtained by cultivation in SD(His⁻) medium at 30° C. for 5 to7 days.

[0290] Although the GWT1-deficient strain shows very slow growth, it wassuggested that the growth is not influenced by the aforementionedcompound (Ia), and the GWT1 gene product is the target of the compound.Furthermore, the GWT1-deficient strain indicated the followingcharacteristics: it cannot grow at high temperatures; the cells areswollen; and in the observation by a transmission electron microscope,the flocculent fibrous structure of the outermost layer of the fungalcell having high electron density had disappeared.

Example A12 Activity of the Aforementioned Compound (Ia) in S.cerevisiae Overexpressing the GWT1 Gene Product

[0291] Using S. cerevisiae CW63 strain and GWT1 gene introduced S.cerevisiae CW63/GWT1, activity of the aforementioned compound (Ia) wasexamined by a method according to the method described in Example A2.

[0292] As a result, even at a concentration (0.39 to 1.56 μg/ml) of theaforementioned compound (Ia) at which cephalosporinase activity in theculture supernatant fraction is increased, and the activity in the cellwall fraction is decreased in S. cerevisiae CW63 strain, no influencewas observed in the S. cerevisiae CW63/GWT1 strain, and even at aconcentration (>3.13 μg/ml) of the aforementioned compound (Ia) at whichgrowth is inhibited in S. cerevisiae CW63 strain, growth inhibition wasnot observed in the S. cerevisiae CW63/GWT1 strain (FIG. 6).

Example A13 Synthesis of (4-Butylphenyl)(1-isoquinolyl)ketone

[0293] Under a nitrogen atmosphere, 1-bromo-4-butylbenzene (2.29 ml,13.0 mmol) was added to a mixed solution of magnesium (338 mg, 13.9mmol) and tetrahydrofuran (6.5 ml), and as an initiator, catalyticamount of 1,2-dibromoethane was added, and this was stirred under refluxfor 10 minutes. The solution was cooled to 0° C., a tetrahydrofuransolution of 1-isoquinolinecarbonitrile (1.0 g, 6.49 mmol) was added, andwas stirred for another 1 hour at room temperature, and at 70° C. for 3hours. Subsequently, the solution was cooled again to 0° C.,concentrated hydrochloric acid (2.56 ml) and methanol (11 ml) wereadded, and then refluxed for 2 hours. The concentrated residue wasdissolved in 5 N sodium hydroxide and toluene, and was filtered throughcelite. The toluene layer of the filtrate was divided, washed withwater, dried over magnesium sulfate, and concentrated. The residue waspurified by silica gel column chromatography to give 1.72 g of the titlecompound.

[0294]¹H-NMR (CDCl₃) δ (ppm): 0.93 (3H, t), 1.32-1.43 (2H, m), 1.58-1.66(2H, m), 2.68 (2H, t), 7.28 (2H, d), 7.61 (1H, td), 7.74 (1H, td), 7.80(1H, d), 7.87 (2H, d), 7.92 (1H, d), 8.20 (1H, d), 8.60 (1H, d).

Example A14 Synthesis of {1-(4-Butylbenzyl)isoquinoline}, theAforementioned Compound of the Formula (Ia)

[0295] The compound of Example A13 (1.72 g, 5.95 mmol), hydrazinemonohydrate (836 mg, 16.7 mmol), and potassium hydroxide (769 mg, 13.7mmol) were added to diethylene glycol (8.5 ml), and were stirred at 80°C. for 1 hour, at 160° C. for 3 and a half hours, and at 200° C. for 1hour. Upon cooling to room temperature, ice water was added andextracted with ethyl acetate. This was washed with water, then driedover magnesium sulfate, and concentrated. The residue was purified bysilica gel column chromatography to give 914 mg of the aforementionedcompound of the formula (Ia).

[0296]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.26-1.36 (2H, m), 1.50-1.59(2H, m), 2.53 (2H, t), 4.64 (2H, s), 7.06 (2H, d), 7.19 (2H, d), 7.53(1H, td), 7.56 (1H, d), 7.64 (1H, td), 7.81 (1H, d), 8.18 (1H, dd,),8.50 (1H, d).

Example A15 Another Method for Producing{1-(4-Butylbenzyl)isoquinoline}, the Aforementioned Compound of theFormula (Ia)

[0297] To a dimethylformamide (1.8 ml) solution of 60% sodium hydride(16 mg, 0.40 mmol), a dimethylformamide (3.6 ml) solution of1-cyano-2-benzoyl-1,2-dihydroisoquinoline (100 mg, 0.38 mmol)synthesized according to the literature of Org. Synth., VI, 115 (1988),and 4-n-butylbenzylchloride (70 mg, 0.38 mmol) was added dropwise undernitrogen atmosphere at −16° C., and was further stirred at roomtemperature for 30 minutes. Water was added, this was concentrated, andtoluene and water were added to this residue. The toluene layer waswashed with water, dried over potassium carbonate, and concentrated. Toan ethanol (1.6 ml) solution of the residue, 50% aqueous sodiumhydroxide solution (0.63 ml) was added, and this was refluxed for 2hours. After concentration, toluene and water were added. The toluenelayer was washed with water, then dried over calcium carbonate, and thenconcentrated. The residue was purified by silica gel columnchromatography to give 18 mg of the aforementioned compound of theformula (Ia).

Example A16 Cloning of the C. albicans Homologue of the S. cerevisiaeGWT1 Gene

[0298] The C. albicans genomic DNA (25 μg) treated with HindIII (TaKaRa)for 16 hours was separated by 0.75% agarose gel electrophoresis method,and the DNA fragments ranging in size from approximately 3.5 to 4.5 kbwere recovered from the gel. The recovered DNA fragments were insertedinto the HindIII site of the pKF3 vector (TaKaRa), and a Candida genomiclibrary was produced.

[0299] Using the produced library, approximately 10,000 colonies weredisplayed on an LB/Ampicillin plate, colony lifting was performed usinga Colony/Plaque Screen (NEN) membrane, and then this was subjected tohybridization. A probe was produced by labeling 20 ng of theapproximately 1.5 kb DNA fragment of SEQ ID NO: 1 with alpha ³³P-dCTP bythe random primer method, and purifying using a GeneQuant column(Amersham-Pharmacia).

[0300] Hybridization was carried out by pre-incubating the membrane in aPerfectHyb™ (TOYOBO) solution at 65° C. for 1 hour, then adding thelabeled probe mentioned above, and incubating further at 65° C. for 2.5hours. Washing was carried out with (i) 2×SSC, 0.05% SDS solution at 25°C. for 5 minutes, (ii) 2×SSC, 0.05% SDS solution at 25° C. for 15minutes, and (iii) 0.1×SSC, 0.1% SDS solution at 50° C. for 20 minutes.The washed membrane was wrapped with Saran Wrap, contacted with an X-RAYFILM (KONICA) for 24 hours at room temperature, and then developed. TheE. coli colonies corresponding to the exposed spots were isolated, andwere subjected to secondary screening. Approximately 200 of the isolatedcolonies were displayed on each LB/Ampicillin plate, colony lifting wasperformed in a similar manner to primary screening, which was followedby hybridization. The conditions for hybridization were the same as theconditions for primary screening.

[0301] As a result, a single colony of E. coli that reacts strongly withthe probe was isolated. Plasmids were collected from this colony, andwhen the contained sequence was determined, a novel sequence having thesame sequence as that revealed in Example A9 (SEQ ID NO: 5) was found(the sequence of Candida GWT1), and was presumed to be a C. albicanshomologue.

Example A17 The S. Pombe Homologue of the S. cerevisiae GWT1 Gene

[0302]S. Pombe genes that show homology to the S. cerevisiae GWT1 gene(SEQ ID NO: 27, and the amino acid sequence of the gene product thereof:SEQ ID NO: 28) were found from a database search, and were considered tobe the S. Pombe homologues of GWT1.

Example A18 Cloning of the Aspergillus fumigatus Homologue of the S.cerevisiae GWT1 Gene

[0303] By genetic sequence analysis, the inventors discovered two highlyconserved regions in the protein encoded by the GWT1 genes of S.cerevisiae, S. pombe, and C. albicans (FIG. 7). Based on the presumedDNA that encodes the amino acid sequence of this conserved region,primers of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31 weredesigned. PCR amplification was carried out using 1 ∥l of the librarypurchased from STRATAGENE (Aspergillus fumigatus cDNA library: #937053)as a template, and using primers of SEQ ID NO: 29 and SEQ ID NO: 31.Furthermore, as a result of carrying out nested-PCR using 1 μg of thisamplified sample as a template, and using primers of SEQ ID NO: 29 andSEQ ID NO: 30, amplification of a single fragment of approximately 250bp was confirmed. When the sequence of this fragment was determined, anovel sequence having homology to the GWT1 gene of S. cerevisiae, shownin SEQ ID NO: 32, was obtained, and this was presumed to be thehomologue of A. fumigatus.

[0304] To obtain a full length cDNA, primers of SEQ ID NO: 33 and SEQ IDNO: 34 were designed based on the sequence of the amplified fragment.Furthermore, primers outside the gene insertion site of the library, SEQID NO: 35 and SEQ ID NO: 36, were designed. As a result of performingPCR using the A. fumigatus cDNA library as a template, and the primerset of SEQ ID NO: 33 and SEQ ID NO: 35, or the primer set of SEQ ID NO:34 and SEQ ID NO: 36, amplification of a DNA fragment of approximately 1kb was confirmed (by both primer sets). As a result of determining thenucleotide sequences of these fragments, a novel sequence that is highlyhomologous to the GWT1 genes of S. cerevisiae shown in SEQ ID NO: 1 wasobtained. Since the sequence is highly homologous to the GWT1 genes ofS. cerevisiae, S. pombe, and C. albicans throughout the entire gene,this sequence was strongly suggested to be a homologue of A. fumigatus.

[0305] To clone the entire homologue of A. fumigatus, the primer shownin SEQ ID NO: 37 that corresponds to the sequence upstream of theinitiation codon, and the primer of SEQ ID NO: 38 that corresponds tothe sequence downstream of the stop codon were newly designed based onthe obtained sequence. As a result of performing 35 cycles of PCR usingthe A. fumigatus cDNA library (STRATAGENE) and the A. fumigatus genomiclibrary (STRATAGENE) as templates, and primers of SEQ ID NO: 37 and SEQID NO: 38, a single amplified fragment of approximately 1.6 kb wasdetected from both templates. As a result of determining the nucleotidesequence of this fragment by Direct-Sequencing, the nucleotide sequenceshown in SEQ ID NO: 39 was found from the cDNA library, and wassuggested to encode a protein comprising 501 amino acids shown in SEQ IDNO: 40. Furthermore, the nucleotide sequence of SEQ ID NO: 41 was foundfrom the genomic library, and was found to have an intron comprising 77base pairs in one position.

Example A19 Cloning of the Cryptococcus Homologue of the S. cerevisiaeGWT1 Gene

[0306] 1). Database Search

[0307] As a result of database searching for genes showing homology tothe S. cerevisiae GWT1 gene, the sequence of 502042C05.x1 was found fromthe server of the Genome Center at Stanford University(http://baggage.stanford.edu/cgi-misc/cneoformans/). Furthermore, thesequence of b6e06cn.f1 was found from the server at Oklahoma University,U.S.A (http://www.genome.ou.edu/cneo_blast.html).

[0308] 2). PCR Using Genomic DNA as Template

[0309] The primer of SEQ ID NO: 42 was constructed based on the sequenceof 502042C05.x1, and the primer of SEQ ID NO: 43 was constructed basedon the sequence of b6e06cn.f1. When PCR amplification was carried outusing the genomic DNA of Cryptococcus (Cryptococcus neoformans) as atemplate, and using the primer of SEQ ID NO: 42, and the primer of SEQID NO: 43, an amplified fragment of approximately 2 kb was detected.When the nucleotide sequence of this fragment was determined, a novelsequence showing homology to the GWT1 gene of S. cerevisiae, shown inSEQ ID NO: 44, was obtained.

[0310] In order to obtain the sequence upstream of the initiation codonof the Cryptococcus GWT1 gene, the primer of SEQ ID NO: 45 was designedbased on the sequence of 502042C05.x1, and the primer of SEQ ID NO: 46was designed based on the sequence of SEQ ID NO: 44. When PCRamplification was carried out using the genomic DNA of Cryptococcus as atemplate, and using the primer of SEQ ID NO: 45, and the primer of SEQID NO: 46, an amplified fragment of approximately 500 bp was detected.When the nucleotide sequence of this fragment was determined, thesequence of SEQ ID NO: 47 was obtained, and this was found to overlapwith SEQ ID NO: 44.

[0311] 3). 3′-RACE

[0312] To obtain the 3′-terminal sequence of the Cryptococcus GWT1 gene,3′-RACE was carried out. Reverse transcription was carried out bypriming with the adaptor-primer of SEQ ID NO: 48, which is based on 16μg of total RNA extracted from Cryptococcus, and by using SuperScript IIReverse Transcriptase (GIBCO/BRL), and a single stranded cDNA, which isto become the template for the RT-PCR that follows, was produced. As aresult of performing 35 cycles of PCR using the single stranded cDNA asa template, and the primers of SEQ ID NO: 49 and SEQ ID NO: 50, anamplified fragment of approximately 1.2 kb was detected. When thenucleotide sequence of this fragment was analyzed by theDirect-Sequencing method, the novel sequence shown in SEQ ID NO: 51showing homology to the S. cerevisiae GWT1 gene was obtained.

[0313] 4). PCR of a Full Length Genomic DNA

[0314] Using the primer of SEQ ID NO: 52 that was designed based on SEQID NO: 47, and the primer of SEQ ID NO: 53 that was designed based onSEQ ID NO: 51, 35 cycles of PCR was carried out on three independentpreparations with the genomic DNA of Cryptococcus as template. As aresult, an amplified fragment of approximately 2 kb was detected fromall three of the independent tubes, and therefore, each of them wereindividually subjected to Direct-Sequencing, and their entire nucleotidesequences were determined. As a result, the three independent sequencescompletely matched, and a sequence comprising the full length GWT1 genehomologue of Cryptococcus shown in SEQ ID NO: 54 was obtained.

[0315] 5). Determination of the cDNA Sequence

[0316] Comparison of the sequence of the Cryptococcus GWT1 gene derivedfrom the genome shown in SEQ ID NO: 54 with cDNA sequence 51 obtained by3′-RACE suggested the presence introns at two positions. Furthermore,since the open reading frame following the ATG initiation codon is notcontinuous, the presence of another intron was suggested. Therefore, thecDNA structure was predicted from the presumed amino acid sequence andthe splicing donor/acceptor sequence, and the primers of SEQ ID NO: 55and SEQ ID NO: 56 were designed at the position predicted to be thejunction between exons. As a result of performing 35 cycles of PCR usingthe single stranded cDNA derived from Cryptococcus as template with theabove-mentioned primers, an amplified fragment of approximately 1.4 kbwas confirmed. As a result of determining the nucleotide sequence bysubjecting the fragment to Direct-Sequencing, the sequence of SEQ ID NO:57 was obtained, and by comparing with SEQ, ID NO: 54, the cDNA sequenceof the GWT1 gene of Cryptococcus was suggested to have the structure ofSEQ ID NO: 58. Since the sequence shows high homology at certain regionswith the GWT1 genes of S. cerevisiae, S. pombe, C. albicans, and A.fumigatus, this sequence was strongly suggested to be a homologue ofCryptococcus.

Example A20 Genetic Mutation That Confers Resistance to theAforementioned Compound of the Formula (Ia)

[0317]S. cerevisiae LW63 strain having a lysozyme gene as the reportergene due to introduction of pRLW63T was treated with ethylmethanesulfonate, then by culturing in a SD medium containing theaforementioned compound of the formula (Ia) at concentrations of 1.56,3.13, and 6.25 μg/ml at 37° C. for 3 days, five resistant mutant strains(R1 to R5) were obtained. Among them, the R1 mutant strain and the R5mutant strain were found to have acquired a specific resistantcharacteristic to the aforementioned compound of the formula (Ia) due toa mutation of a single gene. To confirm whether or not these two mutantstrains have mutations on the GWT1 gene, genomic DNAs were extractedfrom both mutant strains, and the nucleotide sequence of the GWT1 geneportion was determined. As a result, in the R1 mutant strain, guanine atposition 1213 had been mutated to adenine. Furthermore, in the R5 mutantstrain, guanine at position 418 had been mutated to adenine. Therefore,it was elucidated that in the R1 mutant strain, the 405th amino acid,isoleucine, had been changed to valine, and in the R5 mutant strain, the140th amino acid, glycine, had been changed to arginine.

[0318] Next, to confirm whether or not these mutations are the cause ofthe acquisition of the specific resistant characteristic to theaforementioned compound of the formula (Ia), the mutant GWT1 gene (R1 orR5) was isolated using the genomic DNAs derived from both mutant strainsas templates and the primers of SEQ ID NOS: 60 and 61. Simultaneously,the GWT1 promoter region (SEQ ID NO: 62) and the terminator region (SEQID NO: 63) were isolated, the GWT1 gene promoter, mutant GWT1 gene ORF,and the GWT1 gene terminator were inserted into the pRS316 vector, andplasmids that express a single copy of the mutant GWT1 gene wereconstructed (pRS316GWT1-R1, pRS316GWT1-R5). This was introduced to adiploid strain (WDG1) in which only a single copy of the GWT1 gene isdisrupted. Spores were formed by culturing the colonies on a sporulationmedium, and a clone in which the GWT1 gene on the chromosome isdisrupted and also harbors the abovementioned plasmid was obtained byperforming a tetrad analysis. When this was cultured in a mediumcontaining the aforementioned compound of the formula (Ia), resistanceto the aforementioned compound of the formula (Ia) was seen, similarlyto the original R1 mutant strain and R5 mutant strain. From the above,it was elucidated that the specific resistant characteristic to theaforementioned compound of the formula (Ia) is conferred by a pointmutation accompanying an amino acid mutation, that occurred on the GWT1gene, and this compound was strongly suggested to inhibit the functionof the GWT1 protein by directly binding to the protein.

Example B

[0319] The compounds of this invention can be produced, for example, bythe method of the Examples below. However, the Examples are forillustration purpose only and the compounds of this invention are not tobe construed as being limited to those prepared in the followingspecific examples under any circumstances.

Example B1 1-(Chloromethyl)-4-n-butylbenzene

[0320]

[0321] Thionyl chloride (2.5 ml, 34 mmol) was added to a solution of4-n-butylbenzyl alcohol (2.0 g, 12 mmol) in ether (25 ml), and thismixture was stirred at room temperature for 3 hours. After concentrationof the mixture, excess thionyl chloride was removed by azeotropicdistillation with benzene to give the title compound (2.3 g). Thiscompound was used in the following reaction without purification.

Example B2 1-(4-Butylbenzylisoquinoline

[0322]

[0323] A solution of 1-cyano-2-benzoyl-1,2-dihydroisoquinoline (100 mg,0.38 mmol), which was synthesized according to Org. Synth., VI, 115(1988), and 4-n-butylbenzyl chloride (70 mg, 0.38 mmol) indimethylformamide (3.6 ml) was added dropwise to a solution of 60%sodium hydride (16 mg, 0.40 mmol) in dimethylformamide (1.8 ml) undernitrogen atmosphere at −16° C., and this mixture was stirred at roomtemperature for 30 minutes. Water was added, the mixture wasconcentrated under reduced pressure, and toluene and water were added tothe residue. The toluene layer was washed with water, dried overpotassium carbonate, then concentrated under reduced pressure. A 50%aqueous sodium hydroxide solution (0.63 ml) was added to a solution ofthe residue in ethanol (1.6 ml) This mixture was heated under reflux for2 hours and concentrated, and then toluene and water were added. Thetoluene layer was washed with water, dried over calcium carbonate, andthen concentrated under reduced pressure. The residue was purified bysilica gel column chromatography to give the title compound (18 mg).

[0324]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.26-1.36 (2H, m), 1.50-1.59(2H, m), 2.53 (2H, t), 4.64 (2H, s), 7.06 (2H, d), 7.19 (2H, d), 7.53(1H, td), 7.56 (1H, d), 7.64 (1H, td), 7.81 (1H, d), 8.18 (1H, dd), 8.50(1H, d).

Example B3 (4-Butylphenyl)(1-isoquinolyl)ketone

[0325]

[0326] 1-Bromo-4-butylbenzene (2.29 ml, 13 mmol) and a catalytic amountof 1,2-dibromoethane as an initiator were added to a mixed solution ofmagnesium (338 mg, 14 mmol) and tetrahydrofuran (6.5 ml) under nitrogenatmosphere, and this mixture was stirred under reflux for 10 minutes.The mixture was cooled to 0° C., a solution of1-isoquinolinecarbonitrile (1.0 g, 6.5 mmol) in tetrahydrofuran wasadded, and this mixture was stirred at room temperature for 1 hour, thenat 70° C. for 3 hours. Thereafter, the mixture was cooled again to 0°C., concentrated hydrochloric acid (2.6 ml) and methanol (11 ml) wereadded, and this mixture was heated under reflux for 2 hours. After themixture was concentrated, the residue was dissolved in 5 N sodiumhydroxide and toluene, and was filtered through celite. The toluenelayer of the filtrate was separated, washed with water, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (1.7 g).

[0327]¹H-NMR (CDCl₃) δ (ppm): 0.93 (3H, t), 1.32-1.43 (2H, m), 1.58-1.66(2H, m), 2.68 (2H, t), 7.28 (2H, d), 7.61 (1H, td), 7.74 (1H, td), 7.80(1H, d), 7.87 (2H, d), 7.92 (1H, d), 8.20 (1H, d), 8.60 (1H, d).

Example B4 Alternative Method for the Production of1-(4-Butylbenzyl)isoquinoline

[0328] The compound of Example B3 (1.7 g, 6.0 mmol), hydrazinemonohydrate (836 mg, 17 mmol), and potassium hydroxide (769 mg, 14 mmol)were added to diethylene glycol (8.5 ml), and this mixture was stirredat 80° C. for 1 hour, at 160° C. for 3.5 hours, then at 200° C. for 1hour. The mixture was cooled to room temperature, ice water was added,and this was extracted with ethyl acetate. The extract was washed withwater, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (914 mg).

[0329]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.26-1.36 (2H, m), 1.50-1.59(2H, m), 2.53 (2H, t), 4.64 (2H, s), 7.06 (2H, d), 7.19 (2H, d), 7.53(1H, td), 7.56 (1H, d), 7.64 (1H, td), 7.81 (1H, d), 8.18 (1H, dd), 8.50(1H, d).

Example B5 1-(4-Ethylbenzyl)isoquinoline

[0330]

[0331] Using p-ethylbenzyl chloride, the title compound was obtained inthe same manner as in Example B2.

[0332]¹H-NMR (CDCl₃) δ (ppm): 1.18 (3H, t), 2.57 (2H, q), 4.64 (2H, s),7.08 (2H, d), 7.20 (2H, d), 7.50-7.55 (2H, m), 7.61-7.65 (1H, m), 7.80(1H, d), 8.16-8.18 (1H, m), 8.49 (1H, d).

Example B6 (4-Propylphenyl)methanol

[0333]

[0334] A solution of sodium borohydride (2.9 g, 76 mmol) andconcentrated sulfuric acid in ether (prepared by adding 2.0 ml ofconcentrated sulfuric acid to 4.0 ml of ether) was added dropwise to asolution of p-n-propylbenzoic acid (5.0 g, 32 mmol) in tetrahydrofuran(20 ml) cooled to 0° C. keeping the temperature of the reaction systembelow 20° C., and then this mixture was stirred at room temperature for3 hours. After the mixture was cooled on ice, methanol and 1 N sodiumhydroxide were added, and this mixture was extracted with ethyl acetate.The ethyl acetate layer was washed with saturated brine, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure to give the title compound (4.33 g). This compound was used inthe following reaction without purification.

Example B7 1-(Chloromethyl)-4-propylbenzene

[0335]

[0336] The title compound was obtained by treating the compound ofExample B6 in the same manner as in Example B1. This compound was usedin the following reaction without further purification.

Example B8 1-(4-Propylbenzyl)isoquinoline

[0337]

[0338] The title compound was obtained by treating the compound ofExample B7 in the same manner as in Example B2.

[0339]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.55-1.61 (2H, m), 2.51 (2H,t), 4.64 (2H, s), 7.06 (2H, d), 7.19 (2H, d), 7.51-7.55 (2H, m),7.61-7.65 (1H, m), 7.81 (1H, d), 8.17 (1H, dd), 8.49 (1H, d).

Example B9 (4-Pentylphenyl)methanol

[0340]

[0341] The title compound was obtained by reducing 4-n-amylbenzoic acidin the same manner as in Example B6.

Example B10 1-(Chloromethyl)-4-pentylbenzene

[0342]

[0343] The title compound was obtained by treating the compound ofExample B9 in the same manner as in Example B1. This compound was usedin the following reaction without further purification.

Example B11 1-(4-Pentylbenzyl)isoquinoline

[0344]

[0345] The title compound was obtained by treating the compound ofExample B10 in the same manner as in Example B2.

[0346]¹H-NMR (CDCl₃) δ (ppm): 0.86 (3H, t), 1.26-1.33 (4H, m), 1.52-1.59(2H, m), 2.52 (2H, t), 4.64 (2H, s), 7.06 (2H, d), 7.18 (2H, d),7.50-7.55 (2H, m), 7.61-7.65 (1H, m), 7.80 (1H, d), 8.17(1H, dd), 8.49(1H, d).

Example B12 (4-Hexylphenyl)methanol

[0347]

[0348] The title compound was obtained by reducing 4-n-hexylbenzoic acidin the same manner as in Example B6. This compound was used in thefollowing reaction without further purification.

Example B13 1-(Chloromethyl)-4-hexylbenzene

[0349]

[0350] The title compound was obtained by treating the compound ofExample B12 in the same manner as in Example B1. This compound was usedin the following reaction without further purification.

Example B14 1-(4-Hexylbenzyl)isoquinoline

[0351]

[0352] The title compound was obtained by treating the compound ofExample B13 in the same manner as in Example B2.

[0353]¹H-NMR (CDCl₃) δ (ppm): 0.86 (3H, t), 1.26-1.31 (6H, m), 1.51-1.58(2H, m), 2.52 (2H, t), 4.63 (2H, s), 7.06 (2H, d), 7.18 (2H, d),7.50-7.55 (2H, m), 7.61-7.65 (1H, m), 7.80 (1H, d), 8.17 (1H, dd), 8.49(1H, d).

Example B15 1-(4-Isopropylbenzyl)isoquinoline

[0354]

[0355] The title compound was obtained by treating p-isopropylbenzylchloride in the same manner as in Example B2.

[0356]¹H-NMR (CDCl₃) δ (ppm): 1.19 (6H, d), 2.80-2.87 (1H, m), 4.64 (2H,s), 7.11 (2H, d), 7.21 (2H, d), 7.51-7.56 (2H, m), 7.61-7.65 (1H, m),7.81 (1H, d), 8.19 (1H, dd), 8.50 (1H, d).

Example B16 1-[4-(tert-Butyl)benzyl]isoquinoline

[0357]

[0358] The title compound was obtained by treating 4-tert-butylbenzylchloride in the same manner as in Example B2.

[0359]¹H-NMR (CDCl₃) δ (ppm): 1.26 (9H, s), 4.64 (2H, s), 7.22 (2H, d),7.27 (2H, d), 7.52-7.56 (2H, m), 7.62-7.66 (1H, m), 7.81 (1H, d), 8.19(1H, dd), 8.50 (1H, d).

Example B17 (4-Isobutylphenyl)methanol

[0360]

[0361] The title compound was obtained by reducing 4-isobutylbenzoicacid in the same manner as in Example B6. This was used in the followingreaction without further purification.

Example B18 1-(Chloromethyl)-4-isobutylbenzene

[0362]

[0363] The title compound was obtained by treating the compound ofExample B17 in the same manner as in Example B1. This was used in thefollowing reaction without further purification.

Example B19 1-(4-Isobutylbenzyl)isoquinoline

[0364]

[0365] The title compound was obtained by treating the compound ofExample B18 in the same manner as in Example B2.

[0366]¹H-NMR (CDCl₃) δ (ppm): 0.86 (6H, d), 1.75-1.83 (1H, m), 2.39 (2H,d), 4.66 (2H, s), 7.02 (2H, d), 7.18 (2H, d), 7.52-7.58 (2H, m),7.63-7.67 (1H, m), 7.82 (1H, d), 8.18 (1H, d), 8.50 (1H, d).

Example B20 1-(Chloromethyl)-4-(trifluoromethyl)benzene

[0367]

[0368] The title compound was obtained by treating4-trifluoromethylbenzyl alcohol in the same manner as in Example B1.This was used in the following reaction without further purification.

Example B21 1-[4-(Trifluoromethyl)benzyl]isoquinoline

[0369]

[0370] The title compound was obtained by treating the compound ofExample B20 in the same manner as in Example B2.

[0371]¹H-NMR (CDCl₃) δ (ppm): 4.73 (2H, s), 7.39 (2H, d), 7.51 (2H, d),7.54-7.60 (2H, m), 7.65-7.69 (1H, m), 7.84 (1H, d), 8.09-8.10 (1H, m),8.51 (1H, d).

Example B22 1-(Chloromethyl)-4-(trifluoromethoxy)benzene

[0372]

[0373] The title compound was obtained by treating4-trifluoromethoxybenzyl alcohol in the same manner as in Example B1.This was used in the following reaction without further purification.

Example B23 1-[4-(Trifluoromethoxy)benzyl]isoquinoline

[0374]

[0375] The title compound was obtained by treating the compound ofExample B22 in the same manner as in Example B2.

[0376]¹H-NMR (CDCl₃) δ (ppm): 4.67 (2H, s), 7.10 (2H, d) 7.27 (2H, d),7.54-7.59 (2H, m), 7.64-7.68 (1H, m), 7.84 (1H, d), 8.11 (1H, dd), 8.50(1H, d).

Example B24 1-(Chloromethyl)-2-iodobenzene

[0377]

[0378] Methanesulfonyl chloride (2.0 ml, 29 mmol) and triethylamine (3.6ml, 26 mmol) were added to a solution of o-iodobenzyl alcohol (5.0 g, 21mmol) in methylene chloride (50 ml) cooled to 0° C., and the mixture wasstirred at that temperature for 19 hours. A 5% aqueous sodiumhydrogencarbonate solution was added, and the resulting mixture wasextracted with methylene chloride. The methylene chloride layer wasdried over anhydrous magnesium sulfate and concentrated under reducedpressure to give the title compound (5.34 g).

Example B25 1-(2-Iodobenzyl)isoquinoline

[0379]

[0380] The title compound was obtained by treating the compound ofExample B24 in the same manner as in Example B2.

[0381]¹H-NMR (CDCl₃) δ (ppm): 4.74 (2H, s), 6.81-6.84 (1H, m), 6.87-6.92(1H, m), 7.11-7.15 (1H, m), 7.55-7.57 (1H, m), 7.60 (1H, d), 7.64-7.68(1H, m), 7.83-7.86 (1H, m), 7.89-7.91 (1H, m), 8.00-8.02 (1H, m), 8.50(1H, d).

Example B26 1-[2-(2-Phenyl-1-ethynyl)benzyl]isoquinoline

[0382]

[0383] A solution of tetrakis(triphenylphosphine)palladium (58 mg, 0.05mmol) and ethynylbenzene (204 mg, 2.0 mmol) in pyrrolidine (1.5 ml) wasadded to a solution of the compound of Example B25 (345 mg, 1.07 mmol)in pyrrolidine (1.5 ml) under nitrogen atmosphere, and the mixture wasstirred at 80° C. for 3 hours. The mixture was cooled to roomtemperature, diluted with ethyl acetate, washed with a saturated aqueousammonium chloride solution, dried over anhydrous magnesium sulfate, andthen concentrated under reduced pressure. The residue was purified bysilica gel chromatography to give the title compound (280 mg).

[0384]¹H-NMR (CDCl₃) δ (ppm): 4.95 (2H, s), 6.98-7.06 (2H, m), 7.10-7.21(2H, m), 7.31-7.35 (3H, m), 7.48-7.51 (3H, m), 7.57-7.65 (2H, m), 7.82(1H, d), 8.25 (1H, d), 8.52 (1H, d).

Example B27 1-(2-Phenylethylbenzyl)isoquinoline

[0385]

[0386] Palladium-carbon (10%, 230 mg) was added to a solution of thecompound of Example B26 (280 mg, 0.88 mmol) in tetrahydrofuran (30 ml),and this mixture was stirred at room temperature under hydrogenatmosphere (1 atm) for 3 hours. The catalyst was removed by filtrationand the obtained filtrate was concentrated under reduced pressure. Theresidue was purified by silica gel chromatography to give the titlecompound (162 mg).

[0387]¹H-NMR (CDCl₃) δ (ppm): 2.90-2.94 (2H, m), 3.07-3.10 (2H, m), 4.67(2H, s), 6.80 (1H, d), 7.02-7.06 (1H, m), 7.15-7.30 (7H, m), 7.49-7.53(1H, m), 7.58 (1H, d), 7.64-7.68 (1H, m), 7.84 (1H, d), 7.95 (1H, d),8.50 (1H, d).

Example B281-{2-[4-(Tetrahydro-2H-2-pyranyloxy)-1-butynyl]benzyl}-isoquinoline

[0388]

[0389] A solution of tetrakis(triphenylphosphine)palladium (58 mg, 0.05mmol) and 2-(3-butynyloxy)-tetrahydro-2H-pyran (208 mg, 2.0 mmol) inpyrrolidine (1.5 ml) was added to a solution of the compound of ExampleB25 (345 mg, 1.07 mmol) in pyrrolidine (1.5 ml) under nitrogenatmosphere, and this mixture was stirred for four days at roomtemperature, and for another 30 minutes at 80° C. The mixture was cooledto room temperature, diluted with ethyl acetate, washed with a saturatedaqueous ammonium chloride solution, dried over anhydrous magnesiumsulfate, and then concentrated under reduced pressure. The residue waspurified by silica gel chromatography to give the title compound (277mg).

[0390]¹H-NMR (CDCl₃) δ (ppm): 1.42-1.60 (4H, m), 1.64-1.68 (1H, m),1.75-1.81 (1H, m), 2.76-2.80 (2H, m), 3.46-3.51 (1H, m), 3.60-3.66 (1H,m), 3.85-3.95 (2H, m), 4.64-4.66 (1H, m), 4.85 (2H, s), 6.95-6.98 (1H,m), 7.05-7.13 (2H, m), 7.44-7.46 (1H, m), 7.49-7.53 (1H, m), 7.56 (1H,d), 7.60-7.65 (1H, m), 7.80-7.82 (1H, m), 8.15-8.18 (1H, m), 8.49-8.51(1H, m).

Example B29 4-[2-(1-Isoquinolylmethyl)phenyl]-3-butyn-1-ol

[0391]

[0392] After the compound of Example B28 (200 mg, 0.54 mmol) was cooledto 0° C., a hydrochloric acid-methanol solution (10%, 5 ml) was added,and this mixture was stirred for 15 minutes. A saturated aqueous sodiumhydrogencarbonate solution was added, and this mixture was extractedwith ethyl acetate. The ethyl acetate layer was dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The residuewas purified by silica gel chromatography to give the title compound (86mg).

[0393]¹H-NMR (CDCl₃) δ ppm): 2.72 (2H, t), 3.53-3.60 (1H, brs), 3.85(2H, t), 4.85 (2H, s), 7.12-7.15 (2H, m), 7.22-7.24 (1H, m), 7.42-7.44(1H, m), 7.55-7.59 (2H, m), 7.63-7.67 (1H, m), 7.81 (1H, d), 8.30 (1H,m), 8.46 (1H, m).

Example B30 4-[2-(1-Isoquinolylmethyl)phenyl]-1-butanol

[0394]

[0395] Palladium-carbon (10%, 10 mg) was added to a solution of thecompound of Example B29 (44 mg, 0.15 mmol) in tetrahydrofuran (5 ml),and this mixture was stirred at room temperature under hydrogenatmosphere (1 atm) for 1 hour. After the catalyst was removed byfiltration, the filtrate was concentrated under reduced pressure. Theresidue was purified by silica gel chromatography to give the titlecompound (18 mg).

[0396]¹H-NMR (CDCl₃) δ (ppm): 1.61-1.75 (4H, m), 2.33 (1H, brs), 2.77(2H, t), 3.67 (2H, t), 4.70 (2H, s), 6.91 (1H, d), 7.02-7.06 (1H, m),7.12-7.16 (1H, m), 7.19-7.21 (1H, m), 7.50-7.55 (1H, m), 7.57 (1H, d),7.63-7.67 (1H, d), 7.83 (1H, d), 8.09 (1H, d), 8.47 (1H, d).

Example 31 1-Bromo-2-(chloromethyl)benzene

[0397]

[0398] The title compound was obtained by treating p-bromobenzyl alcoholin the same manner as in Example B1.

Example B32 1-(4-Bromobenzyl)isoquinoline 2490

[0399]

[0400] The title compound was obtained by treating the compound ofExample B31 in the same manner as in Example B2.

[0401]¹H-NMR (CDCl₃) δ (ppm): 4.6 1(2H, s), 7.14-7.16 (2H, m), 7.35-7.39(2H, m), 7.52-7.58 (2H, m), 7.63-7.67 (1H, m), 7.82 (1H, d), 8.07-8.10(1H, m), 8.49 (1H, d).

Example B33 Ethyl (E)-3-[4-(Isoquinolylmethyl)phenyl]-2-propanoate

[0402]

[0403] Tris(2-methylphenyl)phosphine (20 mg, 0.067 mmol), palladium(II)acetate (7.5 mg, 0.034 mmol), and triethylamine (70 μl, 0.50 mmol) wereadded to a solution of the compound of Example B32 (100 mg, 0.34 mmol)and vinyl propionate (73 μl, 0.67 mmol) in dimethylformamide (1.0 ml)under nitrogen atmosphere, and this mixture was stirred at 100° C. for 4hours. After the mixture was cooled to room temperature, water wasadded, and this mixture was extracted with ethyl acetate. The organiclayer was washed with water, dried over anhydrous magnesium sulfate, andthen concentrated under reduced pressure. The residue was purified bysilica gel column chromatography to give the title compound (74 mg).

[0404]¹H-NMR (CDCl₃) δ (ppm): 1.32 (3H, t), 4.24 (2H, q), 4.69 (2H, s),6.36 (1H, d), 7.29 (2H, d), 7.42 (2H, d), 7.53-7.67 (4H, m), 7.83 (1H,d), 8.11-8.13 (1H, m), 8.50 (1H, d).

Example B34 Ethyl 3-[4-(1-Isoquinolylmethyl)phenyl]propanoate

[0405]

[0406] Palladium-carbon (10%, 20 mg) was added to a solution of thecompound of Example B33 (71 mg, 0.22 mmol) in methanol (5.0 ml), andthis reaction mixture was stirred at room temperature under hydrogenatmosphere at atmospheric pressure for 5 hours. After the catalyst wasremoved from the reaction mixture by filtration, the filtrate wasconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound (52 mg).

[0407]¹H-NMR (CDCl₃) δ (ppm): 1.20 (3H, t), 2.56 (2H, t), 2.88 (2H, t),4.09 (2H, q), 4.64 (2H, s), 7.09 (2H, d), 7.20 (2H, d), 7.51-7.57 (2H,m), 7.62-7.66 (1H, m), 7.82 (1H, d), 8.15 (1H, dd), 8.50 (1H, d).

Example B35 3-[4-(1-Isoquinolylmethyl)phenyl]-1-propanol

[0408]

[0409] Lithium aluminum hydride (6 mg, 0.16 mmol) was added totetrahydrofuran (1.0 ml) cooled to 0° C. under nitrogen atmosphere. Asolution of the compound of Example B34 (46 mg, 0.14 mmol) intetrahydrofuran (1.0 ml) was further added, and this reaction mixturewas stirred at that temperature for 3 hours. A mixed solution ofmethanol and water (9:1, 1.0 ml) was added to the reaction mixture, asaturated aqueous ammonium chloride solution was further added, thenthis mixture was extracted with chloroform. The organic layer was driedover anhydrous magnesium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (22 mg).

[0410]¹H-NMR (CDCl₃) δ (ppm): 1.30-1.35 (1H, brs), 1.81-1.88 (2H, m),2.64 (2H, t), 3.62-3.65 (2H, m), 4.64 (2H, s), 7.09 (2H, d), 7.20 (2H,d), 7.51-7.57 (2H, m), 7.62-7.66 (1H, m), 7.81 (1H; d), 8.16-8.18 (1H,m), 8.49 (1H, d).

Example 36 1-Isoquinolyl(4-methoxyphenyl)ketone

[0411]

[0412] 4-Bromoanisol (15.3 ml, 122 mmol) and a catalytic amount of1,2-dibromoethane as an initiator were added to a mixed solution ofmagnesium (3059 mg, 125.8 mmol) and tetrahydrofuran (20 ml) undernitrogen atmosphere, and this reaction mixture was stirred while heatingunder reflux for 45 minutes. The mixture was cooled to 0° C., a solutionof 1-isoquinolinecarbonitrile (10.78 g, 69.9 mmol) in tetrahydrofuran(30 ml) was added dropwise thereto, and this reaction mixture wasstirred at room temperature for 2 hours. The reaction mixture was cooledon ice, concentrated hydrochloric acid (24 ml) and methanol (120 ml)were added, and this mixture was heated under reflux for 1.5 hours.After cooling on ice, the mixture was adjusted to pH 8 by adding aqueoussodium hydroxide, extracted with ether, washed with saturated brine,dried over anhydrous magnesium sulfate, and then concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (15.87 g).

[0413]¹H-NMR (CDCl₃) δ (ppm): 3.88 (3H, s), 6.95 (2H, d), 7.61 (1H, dd),7.74 (1H, dd), 7.76 (1H, d), 7.85 (2H, d), 8.17 (1H, dd), 8.60 (1H, d).

Example B37 1-Isoquinolyl(4-methoxyphenyl)methanol

[0414]

[0415] Sodium borohydride (1855 mg) was added to an ice-cooled solutionof the compound of Example B36 (8608 mg) in ethanol (170 ml), and thismixture was stirred at room temperature for 35 minutes. Sodiumborohydride (957 mg) was further added, and this reaction mixture wasstirred at 40° C. for 40 minutes. The reaction mixture was concentratedunder reduced pressure, water was added, and this mixture was extractedwith ether. The organic layer was washed with water and saturated brine,dried over anhydrous magnesium sulfate, and then concentrated underreduced pressure. The obtained title compound (7881 mg) was used in thefollowing reaction without further purification.

[0416]¹H-NMR (DMSO-d6) δ (ppm): 3.66 (3H, s), 6.30-6.32 (1H, brs), 6.81(2H, d), 7.28 (2H, d), 7.54 (1H, dd), 7.68 (1H, dd), 7.76 (1H, d), 7.94(1H, d), 8.37 (1H, d), 8.47 (1H, d).

[0417] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B38 1-Isoquinolyl(4-methoxyphenyl)methyl Acetate

[0418]

[0419] Acetic anhydride (20 ml) was added to a solution of the compoundof Example B37 (7881 mg) in pyridine (100 ml), and this reaction mixturewas stirred at 50° C. for 4 hours. The reaction mixture was concentratedunder reduced pressure and subjected to azeotropic distillation withtoluene. The residue was purified by silica gel column chromatography togive the title compound (8.79 g).

[0420]¹H-NMR (CDCl₃) δ (ppm): 2.22 (3H, s), 3.76 (3H, s), 6.84 (2H, d),7.39 (2H, d), 7.54 (1H, dd), 7.56 (1H, s), 7.60 (1H, d), 7.64 (1H, dd),7,82 (1H, d), 8.19 (1H, d), 8.57 (1H, d).

Example B39 1-(4-Methoxybenzyl)isoquinoline

[0421]

[0422] Palladium-carbon (10%, 4.0 g) was added to a solution of thecompound of Example B38 (8.79 g) in methanol (150 ml), and this mixturewas stirred at room temperature under hydrogen atmosphere at atmosphericpressure for 5.5 hours. The catalyst was removed by filtration throughcelite, and the filtrate was concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to give thetitle compound (4.48 g).

[0423]¹H-NMR (CDCl₃) δ (ppm): 3.74 (3H, s), 4.61 (2H, s), 6.79 (2H, d),7.21 (2H, d), 7.53 (1H, dd), 7.56 (1H, d), 7.63 (1H, dd), 7.80 (1H, d),8.16 (1H, d), 8.49 (1H, d).

Example B40 4-(1-Isoquinolylmethyl)phenol

[0424]

[0425] An aqueous hydrobromic acid solution (47%, 40 ml) was added tothe compound of Example B39 (2185 mg), and this reaction mixture washeated under reflux for 14 hours. The reaction mixture was cooled toroom temperature, further cooled on ice, neutralized with a 50% aqueoussodium hydroxide solution, and extracted with ethyl acetate. The ethylacetate layer was washed with water, dried over anhydrous magnesiumsulfate, and then concentrated under reduced pressure. The obtainedpowder was washed with petroleum ether to give the title compound (1822mg).

[0426]¹H-NMR (DMSO-d6) δ (ppm): 4.48 (2H, s), 6.61 (2H, d), 7.07 (2H,d), 7.60 (1H, dd), 7.68 (1H, d), 7.71 (1H, dd), 7.92 (1H, d), 8.27 (1H,d), 8.41 (1H, d), 9.19 (1H, brs).

Example B41 4-(1-Isoquinolylmethyl)phenyl Trifluoromethanesulfonate

[0427]

[0428] Trifluoromethanesulfonic anhydride (0.55 ml) was added dropwiseto an ice-cold solution of the compound of Example B40 (513 mg) inpyridine (10 ml), and this reaction mixture was stirred at thattemperature for 45 minutes. After ice was added, the reaction mixturewas extracted with ether. The organic layer was washed with water andsaturated brine, dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound (546 mg).

[0429]¹H-NMR (CDCl₃) δ (ppm): 4.69 (2H, s), 7.16 (2H, d), 7.35 (2H, d),7.57 (1H, dd), 7,60 (1H, d), 7.68 (1H, dd), 7.85 (1H, d), 8.09 (1H, d),8.50 (1H, d).

Example B42 1-[4-(2-Phenyl-1-ethynyl)benzyl]isoquinoline

[0430]

[0431] Phenylacetylene (53 μl), palladium acetate (9 mg),1,1′-bis(diphenylphosphino)ferrocene (67 mg), copper(I) iodide (3 mg),lithium chloride (20 mg), and triethylamine (50 μl) were added to asolution of the compound of Example B41 (88 mg) in N,N-dimethylformamide (2.0 ml) that had been degassed and placed undernitrogen, and this mixture was stirred at 80° C. for 8 hours. Aftercooling the mixture to room temperature, water was added, and thismixture was extracted with ethyl acetate. The organic layer was washedwith water and saturated brine, dried over anhydrous magnesium sulfate,and then concentrated under reduced pressure. The residue was purifiedby silica gel column chromatography to give the title compound (53 mg).

[0432]¹H-NMR (CDCl₃) 6 (ppm): 4.69 (2H, s), 7.12-7.32 (3H, m), 7.25 (2H,d), 7.42 (2H, d), 7.43-7.52 (2H, m), 7.54 (1H, dd), 7.58 (1H, d), 7.65(1H, dd), 7.83 (1H, d), 8.10 (1H, d), 8.51 (1H, d).

Example B43 1-(4-Phenethylbenzyl)isoquinoline

[0433]

[0434] Palladium-carbon catalyst (10%, 20 mg) was added to a solution ofthe compound of Example B42 (45 mg) in tetrahydrofuran (2 ml), and thismixture was stirred at room temperature under hydrogen atmosphere atatmospheric pressure for 2 hours. The catalyst was removed by filtrationthrough celite, and the filtrate was concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (23 mg).

[0435]¹H-NMR (CDCl₃) δ (ppm): 2.78-2.90 (4H, m), 4.64 (2H, s), 7.07 (2H,d), 7.10-7.20 (5H, m), 7.22 (2H, d), 7.53 (1H, dd), 7.55 (1H, d), 7.63(1H, dd), 7.80 (1H, d), 8.15 (1H, d), 8.49 (1H, d).

Example B44 1-[4-(4-Phenyl-1-butynyl)benzyl]isoquinoline

[0436]

[0437] The title compound was obtained by treating the compound ofExample B41 and 4-phenyl-1-butyne in the same manner as in Example B42.

[0438]¹H-NMR (CDCl₃) δ (ppm): 2.65 (2H, t), 2.88 (2H, t), 4.68 (2H, s),7.12-7.40 (9H, m), 7.50-7.70 (3H, m), 7.80-7.88 (1H, m), 8.00-8.10 (1H,m), 8.48-8.51 (1H, m).

Example B45 1-[4-(4-Phenyl-1-butyl)benzyl]isoquinoline

[0439]

[0440] The title compound was obtained by treating the compound ofExample B44 in the same manner as in Example B43.

[0441]¹H-NMR (CDCl₃) δ (ppm): 1.55-1.80 (4H, m), 2.50-2.65 (4H, m), 4.68(2H, s), 7.00-7.30 (9H, m), 7.52 (1H, dd), 7.56 (1H, d), 7.63 (1H, dd),7.81 (1H, d), 8.15 (1H, d), 8.50 (1H, d).

Example 461-{4-[4-(Tetrahydro-2H-2-pyranyloxy)-1-butynyl]benzyl}-isoquinoline

[0442]

[0443] The title compound was obtained by treating the compound ofExample B41 and 2-(3-butynyloxy)tetrahydro-2H-pyran in the same manneras in Example B42.

[0444]¹H-NMR (CDCl₃) δ (ppm): 1.48-1.90 (6H, m), 2.67 (2H, t), 3.49-3.55(1H, m), 3.60 (1H, dd), 3.65-3.94 (2H, m), 4.66 (2H, s), 4.65-4.70 (1H,m), 7.14-7.20 (2H, m), 7.23-7.30 (2H, m), 7.53 (1H, dd), 7.58 (1H, d),7.65 (1H, dd), 7.82 (1H, d), 8.10 (1H, d), 8.49 (1H, d).

Example B47 4-[4-(1-Isoquinolylmethyl)phenyl]-3-butyn-1-ol

[0445]

[0446] The compound of Example B46 (1048 mg) was dissolved in a 10%hydrochloric acid-methanol solution (50 ml), and this reaction mixturewas stirred at room temperature for 1.5 hours. The reaction mixture wascooled on ice, a saturated aqueous sodium hydrogencarbonate solution wasadded, and the resulting mixture was extracted with ethyl acetate. Theorganic layer was washed with water and saturated brine, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (666 mg).

[0447]¹H-NMR (CDCl₃) δ (ppm): 2.65 (2H, t), 3.77 (2H, t), 4.65 (2H, s),7.18 (2H, d), 7.29 (2H, d), 7.52 (1H, dd), 7.57 (1H, d), 7.64 (1H, dd),7.81 (1H, d), 8.07 (1H, d), 8.49 (1H, d).

[0448] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B48 4-[4-(1-Isoquinolylmethyl)phenyl]-1-butanol

[0449]

[0450] The title compound was obtained by treating the compound ofExample B47 in the same manner as in Example B43.

[0451]¹H-NMR (CDCl₃) δ (ppm): 1.50-1.70 (4H, m), 2.57 (2H, t), 3.62 (2H,t), 4.64 (2H, s), 7.06 (2H, d), 7.18 (2H, d), 7.53 (1H, dd), 7.55 (1H,d), 7.63 (1H, dd), 7.80 (1H, d), 8.16 (1H, d), 8.49 (1H, d).

[0452] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example 49 1-[4-(3-Cyclopentyl-1-propynyl)benzyl]isoquinoline

[0453]

[0454] The title compound was obtained by treating the compound ofExample B41 and 3-cyclopentyl-1-propyne in the same manner as in

Example B42.

[0455]¹H-NMR (CDCl₃) δ (ppm): 1.25-1.35 (2H, m), 1.45-1.70 (6H, m),1.75-1.85 (2H, m), 2.05-2.13 (1H, m), 4.65 (2H, s), 7.17 (2H, d), 7.27(2H, d), 7.51 (1H, dd), 7.56 (1H, d), 7.64 (1H, dd), 7.81 (1H, d), 8.08(1H, d), 8.49 (1H, d).

Example B50 1-[4-(3-Cyclopentylpropyl)benzyl]isoquinoline

[0456]

[0457] The title compound was obtained by treating the compound ofExample B49 in the same manner as in Example B43.

[0458]¹H-NMR (CDCl₃) δ (ppm): 1.25-1.74 (13H, m), 2.49-2.54 (2H, m),4.64 (2H, s), 7.06 (2H, d), 7.18 (2H, d), 7.53 (1H, dd), 7.55 (1H, d),7.63 (1H, dd), 7.80 (1H, d), 8.17 (1H, d), 8.49 (1H, d).

Example B51 4-[4-(1-Isoquinolylmethyl)phenyl]-2-methyl-3-butyn-2-ol

[0459]

[0460] The title compound was obtained by treating the compound ofExample B41 and 2-methyl-3-butyn-2-ol in the same manner as in ExampleB42.

[0461]¹H-NMR (DMSO-d6) δ (ppm): 1.35 (1H, s), 1.40 (6H, s), 4.62 (2H,s), 7.20-7.30 (4H, m), 7.61 (1H, dd), 7.71 (1H, d), 7.69-7.76 (1H, m),7.95 (1H, d), 8.26 (1H, d), 8.42 (1H, d).

Example B52 4-[4-(1-Isoquinolylmethyl)phenyl]-2-methyl-2-butanol

[0462]

[0463] The title compound was obtained by treating the compound ofExample B51 in the same manner as in Example B43.

[0464]¹H-NMR (CDCl₃) δ (ppm): 1.25 (6H, s), 1.70-1.77 (2H, m), 2.60-2.67(2H, m), 4.64 (2H, s), 7.08 (2H, d), 7.19 (2H, d), 7.53 (1H, dd), 7.55(1H, d), 7.63 (1H, dd), 7.80 (1H, d), 8.16 (1H, d), 8.49 (1H, d).

[0465] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B53 1-[4-(3-Methoxy-1-propynyl)benzyl]isoquinoline

[0466]

[0467] The title compound was obtained by treating the compound ofExample B41 and methylpropargyl ether in the same manner as in ExampleB42.

[0468]¹H-NMR (CDCl₃) δ (ppm): 3.42 (3H, s), 4.29 (2H, s), 4.66 (2H, s),7.21 (2H, d), 7.34 (2H, d), 7.54 (1H, dd), 7.58 (1H, d), 7.65 (1H, dd)7.82 (1H, d), 8.10 (1H, d) 8.49 (1H, d).

Example B54 1-[4-(3-Methoxypropyl)benzyl]isoquinoline

[0469]

[0470] The title compound was obtained by treating the compound ofExample B53 in the same manner as in Example B43.

[0471]¹H-NMR (CDCl₃) δ (ppm): 1.78-1.87 (2H, m), 2.06 (2H, t), 3.31 (3H,s), 3.35 (2H, t), 4.64 (2H, s), 7.07 (2H, d), 7.22 (2H, d), 7.53 (1H,dd), 7.55 (1H, d), 7.64 (1H, dd), 7.81 (1H, d), 8.17 (1H, d), 8.49 (1H,d).

Example B55 1-{4-[2-(2-Pyridyl)-1-ethynyl]benzyl}isoquinoline

[0472]

[0473] The title compound was obtained by treating the compound ofExample B41 and 2-ethynylpyridine in the same manner as in Example B42.

[0474]¹H-NMR (CDCl₃) δ (ppm): 4.71 (2H, s), 7.20-7.25 (2H, m), 7.29 (2H,d), 7.48-7.53 (1H, m), 7.51 (2H, d), 7.57 (1H, dd), 7.61 (1H, d), 7.67(1H, dd), 7.85 (1H, d), 8.13 (1H, d), 8.53 (1H, d), 8.59-8.63 (1H, m).

Example B56 1-{4-[2-(2-Pyridyl)ethyl]benzyl}isoquinoline

[0475]

[0476] The title compound was obtained by treating the compound ofExample B55 in the same manner as in Example B43.

[0477]¹H-NMR (CDCl₃) δ (ppm): 2.94-3.06 (4H, m), 4.64 (2H, s), 7.04 (1H,d), 7.09 (1H, dd), 7.09 (2H, d), 7.18 (2H, d), 7.53 (1H, ddd), 7.54 (1H,dd), 7.55 (1H, d), 7.64 (1H, d) 7.81 (1H, d), 8.15 (1H, d), 8.49 (1H,d), 8.53 (1H, dd).

Example B57 1-{4-[2-(3-Pyridyl)-1-ethynyl]benzyl}isoquinoline

[0478]

[0479] The title compound was obtained by treating the compound ofExample B41 and 3-ethynylpyridine in the same manner as in Example B42.¹H-NMR (CDCl₃) δ (ppm): 4.69 (2H, s), 7.27 (2H, d), 7.31 (1H, dd), 7.43(2H, d), 7.55 (1H, dd), 7.59 (1H, d), 7.66 (1H, dd), 7.82 (1H, ddd),7.83 (1H, d), 8.10 (1H, d), 8.51 (1H, d), 8.60 (1H, dd), 8.77 (1H, d).

Example B58 1-{4-[2-(3-Pyridyl)ethyl]benzyl}isoquinoline

[0480]

[0481] The title compound was obtained by treating the compound ofExample B57 in the same manner as in Example B43.

[0482]¹H-NMR (CDCl₃) δ (ppm): 2.80-2.90 (4H, m), 4.65 (2H, s), 7.04 (2H,d), 7.15 (1H, dd), 7.19 (2H, d), 7.39 (1H, dd), 7.54 (1H, dd), 7.56 (1H,d), 7.64 (1H, dd), 7.81 (1H, d), 8.15 (1H, d), 8.40 (1H, d), 8.42 (1H,d), 8.49 (1H, d).

Example B59 N-(2-Propynyl)acetamide

[0483]

[0484] Pyridine (16.3 ml) and acetic anhydride (10.4 ml) were added toan ice-cooled solution of propargylamine (3023 mg) in methylene chloride(30 ml), and this reaction mixture was stirred at room temperature for lhour. The reaction mixture was poured on ice, extracted with ethylacetate, washed successively with 1 N hydrochloric acid, a saturatedaqueous sodium hydrogencarbonate solution, and saturated brine, driedover anhydrous magnesium sulfate, and then filtered through silica gel.The filtrate was concentrated under reduced pressure to give the titlecompound (743 mg). The obtained compound was used in the followingreaction without further purification.

[0485]¹H-NMR (DMSO-d6) δ (ppm): 1.79 (3H, s), 3.07 (1H, t), 3.81 (2H,d), 8.25 (1H, brs).

Example B60 N-{3-[4-(1-Isoquinolylmethyl)phenyl]-2-propynyl}acetamide

[0486]

[0487] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B59 in the same manner as inExample B42.

[0488]¹H-NMR (DMSO-d6) δ (ppm): 1.79 (3H, s), 4.04 (2H, s), 4.61 (2H,s), 7.45-7.68 (4H, m), 7.68-7.75 (2H, m), 7.90-8.00 (1H, m), 8.25-8.38(2H, m), 8.40-8.45 (1H, m).

Example B61 N-{3-[4-(1-Isoquinolylmethyl)phenyl]propyl}acetamide

[0489]

[0490] The title compound was obtained by treating the compound ofExample B60 in the same manner as in Example B43.

[0491]¹H-NMR (CDCl₃) δ (ppm): 1.95 (3H, s), 1.74-1.84 (2H, m), 2.55 (2H,t), 3.25 (2H, dt), 4.68 (2H, s), 7.10 (2H, d), 7.18 (2H, d), 7.20-7.28(1H, m), 7.50-7.58 (2H, m), 7.60-7.68 (1H, m), 7.75-7.85 (1H, m),8.10-8.16 (1H, m), 8.45-8.50 (1H, m).

Example B62 N-(2-Propynyl)methanesulfonamide

[0492]

[0493] Triethylamine (9.77 ml) was added to an ice-cooled solution ofpropargylamine (3023 mg) in methylene chloride (30 ml). After dropwiseaddition of methanesulfonyl chloride (5.19 ml), the reaction mixture wasstirred for 3 hours at that temperature, warmed to room temperature, andfurther stirred for 2 hours. Ice was added to the reaction mixture, theresulting mixture was extracted with ethyl acetate, washed withsaturated brine, dried over anhydrous magnesium sulfate, andconcentrated under reduced pressure. The residue was dissolved inmethanol (120 ml), potassium carbonate (11.7 g) was added, and thisreaction mixture was stirred at room temperature for 3 hours. Thereaction mixture was concentrated under reduced pressure, neutralizedwith dilute hydrochloric acid while cooling on ice, and then extractedwith ethyl acetate. The extract was washed with saturated brine, driedover anhydrous magnesium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (6.67 g).

[0494]¹H-NMR (CDCl₃) δ (ppm): 2.39 (1H, t), 3.10 (3H, s), 3.99 (2H, dd),4.60 (1H, brs).

Example B63N-{3-[4-(1-Isoquinolylmethyl)phenyl]-2-propynyl}-methanesulfonamide

[0495]

[0496] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B62 in the same manner as inExample B42.

[0497]¹H-NMR (DMSO-d6) δ (ppm): 2.97 (3H, s), 4.00 (2H, d), 4.63 (2H,s), 7.25-7.37 (4H, m), 7.57 (1H, t), 7.62 (1H, dd), 7.71 (1H, d), 7.73(1H, dd), 7.94 (1H, d), 8.28 (1H, d), 8.42 (1H, d).

Example B64N-{3-[4-(1-Isoquinolylmethyl)phenyl]propyl}methanesulfonamide

[0498]

[0499] The title compound was obtained by treating the compound ofExample B63 in the same manner as in Example B43.

[0500]¹H-NMR (CDCl₃) δ (ppm): 1.80-1.90 (2H, m), 2.62 (2H, t), 2.89 (3H,s), 3.11 (2H, dt), 4.25 (1H, brs), 4.64 (2H, s), 7.05 (2H, d) 7.20 (2H,d), 7.50 (1H, dd), 7.56 (1H, d), 7.63 (1H, dd), 7.81 (1H, d), 8.15 (1H,d), 8.49 (1H, d).

Example B65 1-{4-[3-(Ethylsulfanyl)-1-propynyl]benzyl}isoquinoline

[0501]

[0502] The title compound was obtained by treating the compound ofExample B41 and propargyl ethyl sulfide in the same manner as in ExampleB42.

[0503]¹H-NMR (CDCl₃) δ (ppm): 1.30 (3H, t), 2.73 (2H, q), 3.47 (2H, s),4.67 (2H, s), 7.20-7.32 (4H, m), 7.52 (1H, dd), 7.57 (1H, d), 7.64 (1H,dd), 7.81 (1H, d), 8.08 (1H, d), 8.49 (1H, d).

Example B66 t-Butyl N-(Propynyl)carbamate

[0504]

[0505] A solution of di-t butyl-dicarbonate (10.84 g) in tetrahydrofuran(20 ml) was added dropwise to an ice-cooled solution of propargylamine(3040 mg) in tetrahydrofuran (20 ml), the temperature of the mixture wasgradually raised to room temperature, and the reaction mixture wasstirred for 20 hours. After water was added, the reaction mixture wasextracted with ethyl acetate, washed with saturated brine, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure to give the title compound (9.34 g). The obtained compound wasused in the following reaction without further purification.

[0506]¹H-NMR (DMSO-d6) δ (ppm): 1.36 (9H, s), 3.04 (1H, t), 3.62-3.70(2H, m), 7.20-7.30 (1H, m).

Example B67 tert-ButylN-{3-[4-(1-Isoquinolylmethyl)phenyl]-2-propynyl}-carbamate

[0507]

[0508] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B66 in the same manner as inExample B42.

[0509]¹H-NMR (CDCl₃) δ (ppm): 1.45 (9H, s), 4.06-4.13 (2H, m), 4.66 (2H,s), 7.19 (2H, d), 7.20-7.28 (1H, m), 7.29 (2H, d), 7.52 (1H, dd), 7.57(1H, d), 7.65 (1H, dd), 7.82 (1H, d), 8.08 (1H, d), 8.49 (1H, d).

Example B68 tert-ButylN-{3-[4-(1-Isoquinolylmethyl)phenyl]propyl}carbamate

[0510]

[0511] The title compound was obtained by treating the compound ofExample B67 in the same manner as in Example B43.

[0512]¹H-NMR (CDCl₃) δ (ppm): 1.43 (9H, s), 1.70-1.81 (2H, m), 2.54-2.60(2H, m), 3.01-3.20 (2H, m), 4.47-4.57 (1H, m), 4.65 (2H, s), 7.07 (2H,d), 7.21 (2H, d), 7.55 (1H, dd), 7.57 (1H, d), 7.65 (1H, dd), 7.83 (1H,d), 8.18 (1H, d), 8.51 (1H, d).

Example B69 3-[4-(1-Isoquinolylmethyl)phenyl]-2-propyn-1-amine

[0513]

[0514] Trifluoroacetic acid (0.3 ml) was added to an ice-cooled solutionof the compound of Example B67 (4 mg) in methylene chloride (0.6 ml),and the reaction mixture was stirred at that temperature for 1 hour.After a saturated aqueous sodium hydrogencarbonate solution was added,the reaction mixture was extracted with ethyl acetate, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (4 mg).

[0515]¹H-NMR (CDCl₃) δ (ppm): 3.60-3.68 (2H, m), 4.66 (2H, s), 7.19 (2H,d), 7.29 (2H, d), 7.53 (1H, dd), 7.56 (1H, d), 7.63 (1H, dd), 7.82 (1H,d), 8.10 (1H, d), 8.49 (1H, d).

[0516] The amine proton was not observed in the NMR spectrum.

Example B70 3-[4-(1-Isoquinolylmethyl)phenyl]-1-propanamine

[0517]

[0518] The title compound was obtained by treating the compound ofExample B68 in the same manner as in Example B69.

[0519]¹H-NMR (CDCl₃) δ (ppm): 1.20-1.30 (2H, m), 1.78-1.88 (2H, m),2.45-2.52 (2H, m), 2.73-2.81 (2H, m), 4.55 (2H, s), 6.94 (2H, d), 7.08(2H, d), 7.50 (1H, dd), 7.51 (1H, d), 7.61 (1H, dd), 7.76 (1H, d), 8.10(1H, d), 8.38 (1H, d).

Example B71 N-Methyl-N-(2-propynyl)acetamide

[0520]

[0521] The title compound was obtained by treatingN-methyl-N-(2-propynyl)amine in the same manner as in Example B59.

[0522]¹H-NMR (CDCl₃) δ (ppm): 2.11 (2.1H, s), 2.17 (0.9H, s), 2.21(0.7H, t), 2.31 (0.3H, t), 3.00 (0.9H, s), 3.08 (2.1H, s), 4.04 (0.6H,d), 4.23 (1.4H, d).

[0523] The obtained compound contained a 7:3 mixture of geometricalisomers of the amide.

Example B72N-{3-[4-(1-Isoquinolylmethyl)phenyl]-2-propynyl}-N-methyl-acetamaide

[0524]

[0525] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B71 in the same manner as inExample B42.

[0526]¹H-NMR (CDCl₃) δ (ppm): 2.10 (1.8H, s), 2.11 (1.2H, s), 3.01(1.2H, s), 3.10 (1.8H, s), 4.21 (1.2H, s), 4.41 (0.8H, s), 4.67 (2H, s),7.18-7.23 (2H, m), 7.29-7.32 (2H, m), 7.53 (1H, dd), 7.58 (1H, d), 7.65(1H, dd), 7.82 (1H, d), 8.09 (1H, d), 8.49 (1H, d).

[0527] The obtained compound contained a 3:2 mixture of geometricalisomers of the amide.

Example B73N-{3-[4-(1-Isoquinolylmethyl)phenyl]propyl}-N1-methylacetamide

[0528]

[0529] The title compound was obtained by treating the compound ofExample B72 in the same manner as in Example B43.

[0530]¹H-NMR (CDCl₃) δ (ppm): 1.70-1.90 (2H, m), 1.89 (1.5H, s), 2.03(1.5H, s), 2.50-2.59 (2H, m), 2.88 (1.5H, s), 2.91 (1.5H, s), 3.20-3.25(1H, m), 3.36-3.40 (1H, m), 4.66 (2H, s), 7.03-7.10 (2H, m), 7.18-7.30(2H, m), 7.53 (1H, dd), 7.58 (1H, d), 7.66 (1H, dd), 7.82 (1H, d), 8.17(1H, d), 8.50 (1H, d).

[0531] The obtained compounds contained a 1:1 mixture of geometricalisomers of the amide.

Example B74 N-Methyl-N-(2-propynyl)methanesulfonamide

[0532]

[0533] Triethylamine (6.55 ml) was added to an ice-cooled solution ofN-methyl-N-(2-propynyl)amine (2603 mg) in methylene chloride (25 ml).Methanesulfonyl chloride (3.50 ml) was further added dropwise, thereaction mixture was stirred at that temperature for 1 hour, and thenstirred further at room temperature for 2 hours. After ice was added,the reaction mixture was extracted with ethyl acetate, washedsuccessively with 1 N hydrochloric acid, a saturated aqueous sodiumhydrogencarbonate solution, and saturated brine, dried over anhydrousmagnesium sulfate, and then filtered through silica gel. The filtratewas concentrated under reduced pressure to give the title compound (4522mg). The obtained compound was used in the following reaction withoutfurther purification.

[0534]¹H-NMR (CDCl₃) δ (ppm): 2.41 (1H, t), 2.93 (3H, s), 2.96 (3H, s),4.09 (2H, d).

Example B75 N-{3-[4-(1-Isoquinolylmethyl)phenyl]-2-propynyl}-N-methylMethanesulfonamide

[0535]

[0536] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B74 in the same manner as inExample B42.

[0537]¹H-NMR (CDCl₃) δ (ppm): 2.95 (3H, s), 2.97 (3H, s), 4.26 (2H, s),4.68 (2H, s), 7.24 (2H, d), 7.31 (2H, d), 7.55 (1H, dd), 7.59 (1H, d),7.66 (1H, dd), 7.83 (1H, d), 8.10 (1H, d), 8.49 (1H, d).

Example B76 N-{3-[4-(1-Isoquinolylmethyl)phenyl]propyl}-N-methylMethanesulfonamide

[0538]

[0539] Treating the compound of Example B75 in the same manner as inExample B43, the obtained residue was separated and purified by LC-MS[eluent: an acetonitrile solution containing 0.1% trifluoroacetic acid:an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)] to give the title compound.

[0540] MS m/z (ESI:MH⁺): 369.2.

Example B77 5-[4-(1-Isoquinolylmethyl)phenyl]-4-pentyn-2-ol

[0541]

[0542] The title compound was obtained by treating the compound ofExample B41 and 4-pentyn-2-ol in the same manner as in Example B42.

[0543]¹H-NMR (CDCl₃) δ (ppm): 1.27 (3H, t), 2.38-2.62 (2H, m), 3.95-4.03(1H, m), 4.65 (2H, s), 7.19 (2H, d), 7.29 (2H, d), 7.52 (1H, dd), 7.57(1H, d), 7.64 (1H, dd), 7.81 (1H, d), 8.08 (1H, d), 8.48 (1H, d).

[0544] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B78 5-[4-(1-Isoquinolylmethyl)phenyl]-2-pentanol

[0545]

[0546] Treating the compound of Example B77 in the same manner as inExample B43, the obtained residue was separated and purified by LC-MS[eluent: an acetonitrile solution containing 0.1% trifluoroacetic acid:an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)] to give the title compound.

[0547] MS m/z(ESI:MH⁺): 306.2.

Example B79 3-Butylphenol

[0548]

[0549] The title compound was obtained by treating1-butyl-3-methoxybenzene in the same manner as in Example B40.

[0550]¹H-NMR (CDCl₃) δ (ppm): 0.94 (3H, t), 1.30-1.55 (2H, m), 1.55-1.62(2H, m), 2.56 (2H, t), 4.76 (1H, brs), 6.63 (1H, dd), 6.66 (1H, d), 6.75(1H, d), 7.12 (1H, dd).

Example B80 1-Butyl-3-(methoxymethoxy)benzene

[0551]

[0552] A 60% suspension of sodium hydride dispersed in mineral oil (102mg) was added to an ice-cooled solution of the compound of Example B79(318 mg) in dimethylformamide (5 ml), and the reaction mixture wasstirred at room temperature for 30 minutes. The mixture was cooled againon ice, chloromethyl methyl ether (0.18 ml) was added, and this reactionmixture was stirred at room temperature for 12 hours. After water wasadded, the reaction mixture was extracted with ethyl acetate, washedwith a saturated aqueous sodium hydrogencarbonate solution and saturatedbrine, dried over anhydrous magnesium sulfate, and then filtered throughsilica gel. The filtrate was concentrated under reduced pressure to givethe title compound (341 mg). The obtained compound was used in thefollowing reaction without further purification.

[0553]¹H-NMR (CDCl₃) δ (ppm): 0.94 (3H, t), 1.30-1.42 (2H, m), 1.55-2.04(2H, m), 2.58 (2H, t), 3.49 (3H, s), 5.17 (2H, s), 6.80-6.87 (3H, m),7.18 (1H, dd).

Example B81 4-Butyl-2-(methoxymethoxy)benzaldehyde

[0554]

[0555] A solution of t-butyl lithium in pentane (1.51 M, 10.6 ml) wasadded dropwise to a solution of the compound of Example B80 (2396 mg) inpetroleum ether cooled to −20° C., and this reaction mixture was stirredat a temperature in the range of −10° C. to 0° C. for 1.5 hours. Thereaction mixture was cooled to −70° C., anhydrous ether (17 ml) anddimethylformamide (1.91 ml) were added, and the resulting mixture wasstirred at that temperature for 3 hours, then stirred for another 1 hourat room temperature. The reaction mixture was cooled on ice, a saturatedaqueous ammonium chloride solution was added, and the mixture wasextracted with ethyl acetate. The extract was washed with saturatedbrine, dried over anhydrous magnesium sulfate, and then concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (1821 mg).

[0556]¹H-NMR (CDCl₃) δ (ppm): 0.94 (3H, t), 1.32-1.42 (2H, m), 1.57-1.65(2H, m), 2.64 (2H, t), 3.54 (3H, s), 5.29 (2H, s), 6.91 (1H, d), 7.01(1H, s), 7.76 (1H, d), 10.44 (1H, s).

Example B82 [4-Butyl-2-(methoxymethoxy)phenyl](1-isoquinolyl)methanol

[0557]

[0558] An aqueous sodium hydroxide solution (50%, 1.4 ml) was added to asolution of 1-cyano-benzoyl-1,2-dihydroisoquinoline (815 mg), which wassynthesized according to Org. Synth., IV, 155 (1988), the compound ofExample B81 (869 mg), and triethylbenzylammonium chloride (7 mg) inmethylene chloride (1.6 ml), and the reaction mixture was subjected toultrasonication in a water bath for 10 minutes. After methylene chloride(8.3 ml) and ethanol (4.4 ml) were added, the reaction mixture wasfurther subjected to ultrasonication in a water bath for 85 minutes.Water was added and the resulting reaction mixture was extracted withmethylene chloride. The extract was dried over anhydrous magnesiumsulfate, then concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compound(1144 mg).

[0559]¹H-NMR (DMSO-d6) δ (ppm): 0.86 (3H, t), 1.22-1.31 (2H, m),1.44-1.52 (2H, m), 2.44-2.51 (2H, m), 3.16 (3H, s), 5.10 (1H, d), 5.12(1H, d), 6.72 (1H, s), 6.75 (1H, d), 6.84 (1H, s), 7.21 (1H, d), 7.61(1H, dd), 7.72 (1H, dd), 7.74 (1H, d), 7.95 (1H, d), 8.31 (1H, d), 8.42(1H, d).

[0560] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B83 [4-Butyl-2-(methoxymethoxy)phenyl](1-isoquinolyl)methylAcetate

[0561]

[0562] The title compound was obtained by treating the compound ofExample B82 in the same manner as in Example B38.

[0563]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.28-1.40 (2H, m), 1.50-1.60(2H, m), 2.22 (3H, s), 2.54 (2H, t), 3.41 (3H, s), 5.22 (1H, d), 5.26(1H, d), 6.77 (1H, d), 6.94 (1H, s), 7.29 (1H, d), 7.55 (1H, dd), 7.58(1H, d), 7.70 (1H, dd), 7.81 (1H, d), 8.05 (1H, s), 8.35 (1H, d), 8.55(1H, d).

Example B84 1-[4-Butyl-2-(methoxymethoxy)benzyl]isoquinoline

[0564]

[0565] The title compound was obtained by treating the compound ofExample B83 in the same manner as in Example B39.

[0566]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.28-1.37 (2H, m), 1.50-1.58(2H, m), 2.53 (2H, t), 3.46 (3H, s), 4.65 (2H, s), 5.24 (2H, s), 6.66(1H, dd), 6.89 (1H, d), 6.92 (1H, d), 7.51 (1H, dd), 7.53 (1H, d), 7.62(1H, dd), 7.79 (1H, d), 8.23 (1H, d), 8.47 (1H, d).

Example B85 5-Butyl-2-(1-isoquinolylmethyl)phenol

[0567]

[0568] 5 N hydrochloric acid (1.0 ml) was added to a solution of thecompound of Example B84 (88 mg) in methanol (1.5 ml), and this reactionmixture was stirred at room temperature for 14 hours. The reactionmixture was neutralized with a 5 N aqueous sodium hydroxide solution,adjusted to pH 6.8 with phosphate buffer, and extracted with ethylacetate. The extract was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure to give the title compound (44 mg).

[0569]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.23-1.37 (2H, m), 1.48-1.60(2H, m), 2.51 (2H, t), 4.56 (2H, s), 6.65 (1H, dd), 6.82 (1H, d),7.21,(H, d), 7.55 (1H, d), 7.68 (1H, dd), 7.72 (1H, dd), 7.82 (1H, d),8.35 (1H, d), 8.44 (1H, d).

[0570] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B86N-{3-[4-(1-Isoquinolylmethyl)phenyl]-2-propynyl}-N,N-dimethylamine

[0571]

[0572] The title compound was obtained by treating the compound ofExample B41 and 1-dimethylamino-2-propyne in the same manner as inExample B42.

[0573]¹H-NMR (CDCl₃) δ (ppm): 2.04 (3H, s), 2.34 (3H, s), 3.47 (2H, s),4.66 (2H, s), 7.20 (2H, d), 7.32 (2H, d), 7.53 (1H, dd), 7.56 (1H, d),7.65 (1H, dd), 7.82 (1H, d), 8.10 (1H, d), 8.50 (1H, d).

Example B871-{4-[3-(Tetrahydro-2H-2-pyranyloxy)-1-propynyl]benzyl}isoquinoline

[0574]

[0575] The title compound was obtained by treating the compound ofExample B41 and tetrahydro-2-(2-propynyloxy)-2H-pyran in the same manneras in Example B42.

[0576]¹H-NMR (CDCl₃) δ (ppm): 1.45-1.85 (6H, m), 3.50-3.60 (1H, m),3.84-3.90 (1H, m), 4.42 (1H, d), 4.48 (1H, d), 4.66 (2H, 8), 4.87 (1H,dd), 7.15-7.21 (2H, m), 7.33-7.36 (2H, m), 7.50-7.70 (3H, m), 7.81-7.86(1H, m), 8.07-8.10 (1H, m), 8.48-8.51 (1H, m).

Example B88 3-[4-(1-Isoquinolylmethyl)phenyl]-2-propyn-1-ol

[0577]

[0578] The title compound was obtained by treating the compound ofExample B87 in the same manner as in Example B47.

[0579]¹H-NMR (CDCl₃) δ (ppm): 1.20-1.30 (1H, m), 4.46 (2H, s), 4.67 (2H,s), 7.23 (2H, d), 7.31 (2H, d), 7.53 (1H, dd), 7.58 (1H, d), 7.65 (1H,dd), 7.83 (1H, d), 8.09 (1H, d), 8.49 (1H, d).

Example B89 N,N-Dimethyl-4-pentynamide

[0580]

[0581] Dimethylamine (2 M solution in tetrahydrofuran, 8.53 ml),triethylamine (2.59 ml), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (3221 mg), were added to asolution of 4-pentynoic acid (552 mg) in methylene chloride (150 ml) andthis reaction mixture was stirred at room temperature for 24 hours. Thereaction mixture was washed successively with 1 N hydrochloric acid, asaturated aqueous sodium hydrogencarbonate solution, water, andsaturated brine, dried over anhydrous magnesium sulfate, thenconcentrated under reduced pressure to give the title compound (129 mg).The obtained compound was used in the following reaction without furtherpurification.

[0582]¹H-NMR (CDCl₃) δ (ppm): 1.96-1.99 (1H, m), 2.50-2.60 (4H, m), 2.96(3H, s), 3.02 (3H, s).

Example B90 N,N-dimethyl-5-[4-(1-isoquinolylmethyl)phenyl]-4-pentynamide

[0583]

[0584] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B89 in the same manner as inExample B42.

[0585]¹H-NMR (CDCl₃) δ (ppm): 2.59-2.64 (2H, m), 2.71-2.75 (2H, m), 2.96(3H, s), 3.03 (3H, s) 4.66 (2H, s), 7.18 (2H, d), 7.28 (2H, d),7.43-7.70 (3H, m), 7.90 (1H, d), 8.09 (1H, d), 8.50 (1H, d).

Example B91 1-Methyl-2-propynyltetrahydro-2H-2-pyranyl Ether

[0586]

[0587] 3,4-Dihydro-2H-pyran (7.15 ml) and pyridinium p-toluenesulfonate(2187 mg) were added to a solution of 3-butyn-2-ol (3051 mg) indichloromethane (150 ml), and this reaction mixture was stirred at roomtemperature for 29 hours.

[0588] The reaction mixture was washed successively with a saturatedaqueous sodium hydrogencarbonate solution, water, and saturated brine,dried over anhydrous magnesium sulfate, and then concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (4698 mg).

[0589]¹H-NMR (CDCl₃) δ (ppm): 1.45 (1.05H, d), 1.48 (1.95H, d),1.50-1.90 (6H, m), 2.37 (0.65H, d), 2.43 (0.35H, d), 3.50-3.60 (1.3H,m), 3.80-3.86 (0.7H, m), 4.4-3-4.50 (0.35H, m), 4.52-4.60 (0.65H, m),4.77 (0.35H, t), 4.94 (0.65H, t).

Example B921-{4-[3-(Tetrahydro-2H-2-pyranyloxy)-1-butynyl]benzyl)isoquinoline

[0590]

[0591] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B91 in the same manner as inExample B42.

[0592]¹H-NMR (CDCl₃) δ (ppm): 1.40-1.80 (6H, m), 1.49 (1.05H, d), 1.52(1.95H, d), 3.49-3.60 (1H, m), 3.80-3.88 (0.65H, m), 3.99-4.06 (0.35H,m), 4.65 (2H, s), 4.74 (1H, q), 4.83 (0.35H, t), 4.97 (0.65H, t),7.18-7.22 (2H, m), 7.32 (2H, d), 7.54 (1H, dd), 7.57 (1H, d), 7.64 (1H,dd), 7.82 (1H, d), 8.08 (1H, d), 8.49 (1H, d).

Example B93 4-[4-(1-Isoquinolylmethyl)phenyl]-3-butyn-2-ol

[0593]

[0594] The title compound was obtained by treating the compound ofExample B92 in the same manner as in Example B47.

[0595]¹H-NMR (CDCl₃) δ (ppm): 1.53 (3H, d), 2.15 (1H, brs), 4.68 (2H,s), 4.72 (1H, q), 7.21 (2H, d), 7.31 (2H, d), 7.54 (1H, dd), 7.59 (1H,d), 7.66 (1H, dd), 7.84 (1H, d), 8.10 (1H, d), 8.51 (1H, d).

Example B94 4-[4-(1-Isoquinolylmethyl)phenyl]-2-butanol

[0596]

[0597] Treating the compound of Example B93 in the same manner as inExample B43, the obtained residue was separated and purified by LC-MS[eluent: an acetonitrile solution containing 0.1% trifluoroacetic acid:an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)] to give the title compound.

[0598] MS m/z(ESI:MH⁺): 292.2.

Example B95 2-Methyl-4-pentyn-2-ol

[0599]

[0600] Lithium acetylide-ethylenediamine complex was added gradually toa mixed solution of isobutylene oxide (1889 mg) in tetrahydrofuran(13ml) and dimethylsulfoxide (20 ml) cooled to 0° C., and this reactionmixture was stirred at 0° C. for 5 hours. After water was added, thereaction mixture was extracted with ethyl acetate, washed with saturatedbrine, dried over anhydrous magnesium sulfate, and then filtered throughsilica gel. The filtrate was concentrated under reduced pressure to givethe title compound (3316 mg). This was used in the following reactionwithout further purification.

[0601]¹H-NMR (CDCl₃) δ (ppm): 1.33 (6H, s), 2.09 (1H, t), 2.38 (2H, t).

[0602] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B96 5-[4-(1-Isoquinolylmethyl)phenyl]-2-methyl-4-pentyn-2-ol

[0603]

[0604] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B95 in the same manner as inExample B42.

[0605]¹H-NMR (DMSO-d6) δ (ppm): 1.18 (6H, s), 2.28 (1H, s), 2.42 (2H,s), 4.62 (2H, s), 7.10-7.30 (4H, m), 7.62 (1H, dd), 7.71 (1H, d), 7.72(1H, dd), 7.94 (1H, d), 8.27 (1H, d), 8.42 (1H, d).

Example B97 5-[4-(1-Isoquinolylmethyl)phenyl]-2-methyl-2-pentanol

[0606]

[0607] Treating the compound of Example B96 in the same manner as inExample B43, the obtained residue was separated and purified by LC-MS[eluent: an acetonitrile solution containing 0.1% trifluoroacetic acid:an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)] to give the title compound.

[0608] MS m/z (ESI:MH⁺): 320.2.

Example B98 4-Benzyloxy-2-(methoxymethoxy)benzaldehyde

[0609]

[0610] N,N-diisopropylethylamine (1.98 ml) and chloromethyl methyl ether(0.76 ml) were added to a solution of 4-benzyloxy-2-hydroxybenzaldehyde(2071 mg) in tetrahydrofuran (30 ml), and this reaction mixture wasstirred and heated under reflux for 19 hours. N,N-diisopropylethylamine(2.7 ml) and chloromethyl methyl ether (1.04 ml) were further added, andthe resulting mixture was stirred and heated under reflux for another 10hours. After water was added, the reaction mixture was extracted withethyl acetate, washed with a saturated aqueous ammonium chloridesolution and saturated brine, dried over anhydrous magnesium sulfate,then filtered through silica gel and alumina. The filtrate wasconcentrated under reduced pressure to give the title compound (2470mg). This compound was used in the following reaction without furtherpurification.

[0611]¹H-NMR (CDCl₃) δ (ppm): 3.52 (3H, s), 5.12 (2H, s), 5.27 (2H, s),6.68 (1H, dd), 6.80 (1H, d), 7.33-7.45 (5H, m), 7.82 (1H, d), 10.33 (1H,s).

Example B99[4-(Benzyloxy)-2-(methoxymethoxy)phenyl](1-isoquinolyl)methanol

[0612]

[0613] The title compound was obtained by treating the compound ofExample B98 in the same manner as in Example B82.

[0614]¹H-NMR (DMSO-d6) δ (ppm): 3.16 (3H, s), 5.01 (2H, s), 5.1 (11H,d), 5.14 (1H, d), 6.59 (1H, dd), 6.66-6.70 (2H, m), 7.18 (1H, d), 7.31(1H, d), 7.34-7.42 (4H, m), 7.61 (1H, dd), 7.71 (1H, d), 7.75 (1H, d),7.95 (1H, d), 8.28 (1H, d), 8.43 (1H, d).

[0615] The proton of the-hydroxyl group was not observed in the NMRspectrum.

Example B100[4-(Benzyloxy)-2-(methoxymethoxy)phenyl](1-isoquinolyl)methyl Acetate

[0616]

[0617] The title compound was obtained by treating the compound ofExample B99 in the same manner as in Example B38.

[0618]¹H-NMR (CDCl₃) δ (ppm): 2.21 (3H, s), 3.42 (3H, s), 4.98 (1H, d),5.00 (1H, d), 5.21-5.27 (2H, m), 6.54 (1H, dd), 6.81 (1H, d), 7.25 (1H,d), 7.30-7.41 (5H, m), 7.53 (1H, dd), 7.57 (1H, d), 7.63 (1H, dd), 7.80(1H, d), 8.00 (1H, s), 8.29 (1H, d), 8.55 (1H, d).

Example B101 4-(1-Isoquinolylmethyl)-3-(methoxymethoxy)phenol

[0619]

[0620] The title compound was obtained by treating the compound ofExample B100 in the same manner as in Example B39.

[0621]¹H-NMR (DMSO-d6) δ (ppm): 3.36 (3H, s), 4.44 (2H, s), 5.17 (2H,s), 6.22 (1H, d), 6.52 (1H, s), 6.67 (1H, d), 7.57-7.76 (3H, m), 7.92(1H, d), 8.22 (1H, d), 8.37 (1H, d), 9.24 (1H, brs).

Example B102 4-(1-Isoquinolylmethyl)-3-(methoxymethoxy)phenylTrifluoromethanesulfonate

[0622]

[0623] The title compound was obtained by treating the compound ofExample B101 in the same manner as in Example B41.

[0624]¹H-NMR (CDCl₃) δ (ppm): 3.43 (3H, s), 4.65 (2H, s), 5.24 (2H, s),6.77 (1H, dd), 7.04 (1H, d), 7.07 (1H, d), 7.54-7.61 (2H, m), 7.67 (1H,dd), 7.84 (1H, d), 8.16 (1H, d), 8.47 (1H, d).

Example B1031-{2-(Methoxymethoxy)-[4-(tetrahydro-2H-2-pyranyloxy)-1-butynyl]benzyl}isoquinoline

[0625]

[0626] The title compound was obtained by treating the compound ofExample B102 and 2-(3-butynyloxy)tetrahydro-2H-pyran in the same manneras in Example B42.

[0627]¹H-NMR (CDCl₃) δ (ppm): 1.51-1.90 (6H, m), 2.68 (2H, t), 3.50 (3H,s), 3.49-3.55 (1H, m), 3.58-3.65 (1H, m), 3.84-3.94 (2H, m), 4.63-4.68(1H, m), 4.65 (2H, s), 5.23 (2H, s), 6.76 (1H, dd), 7.04 (1H, d), 7.07(1H, d), 7.49-7.69 (3H, m), 7.81 (1H, d), 8.14 (1H, d), 8.47 (1H, d).

Example B104 5-(4-Hydroxy-1-butynyl)-2-(1-isoquinolylmethyl)phenol

[0628]

[0629] The title compound was obtained by treating the compound ofExample B103 in the same manner as in Example B85.

[0630]¹H-NMR (CDCl₃) δ (ppm): 1.80 (1H, brs), 2.66 (2H, t), 3.73-3.82(2H, m), 4.58 (2H, s), 6.87 (1H, d), 7.04 (1H, s), 7.23 (1H, d), 7.60(1H, d), 7.69-7.78 (2H, m), 7.86 (1H, d), 8.37 (1H, d), 8.42 (1H, d).

[0631] The proton of the phenolic hydroxyl group was not observed in theNMR spectrum.

Example B105 1-(t-Butyl)-1,1-dimethylsilyl{4-[4-(1-Isoquinolylmethyl)-phenyl]-2-methyl-3-butynyl}ether

[0632]

[0633] Triphenylphosphine (18.37 g) was added to an ice-cooled solutionof carbon tetrabromide (11.19 g) in methylene chloride (60 ml), and thisreaction mixture was stirred at that temperature for 1 hour. A solutionof 3-{[1-(t-butyl)-1,1-dimethylsilyl]oxy}-2-methylpropanal, which wassynthesized according to Tetrahedron Lett., 4347 (1979), in methylenechloride (14 ml) was added dropwise, and the resulting reaction mixturewas further stirred for 1 hour. The reaction mixture was diluted withmethylene chloride, washed successively with saturated aqueous sodiumhydrogencarbonate solution, saturated an aqueous ammonium chloridesolution and saturated brine, dried over magnesium sulfate, and thenconcentrated under reduced pressure. Ether was added to this residue,insoluble material was separated by filtration, and the filtrate wasconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to givet-butyl[(4,4-dibromo-2-methyl-3-butenyl)oxy]-dimethylsilane (2385 mg).

[0634] Next, a 2.47 M n-butyl lithium solution in hexane (3.15 ml) wasadded dropwise to a solution oft-butyl[(4,4-dibromo-2-methyl-3-butenyl)oxy]dimethylsilane (1326 mg) intetrahydrofuran (10 ml) cooled to −70° C., and this mixture was stirredat that temperature for 1 hour. A saturated aqueous ammonium chloridesolution was further added, and the resulting mixture was warmed to roomtemperature. After water was added, the reaction mixture was extractedwith ether. The ether layer was washed with saturated brine, dried overanhydrous magnesium sulfate, then filtered through silica gel. Thefiltrate was concentrated under reduced pressure. The obtained residueand the compound of Example B41 were treated in the same manner as inExample B42 to obtain the title compound.

[0635]¹H-NMR (CDCl₃) δ (ppm): 0.07 (6H, s), 0.90 (9H, s), 1.18 (3H, d),2.70-2.80 (1H, m), 3.47 (1H, dd), 3.70 (1H, dd), 4.65 (2H, s), 7.16 (2H,d), 7.27 (2H, d), 7.51 (1H, dd), 7.56 (1H, d), 7.64 (1H, dd), 7.81 (1H,d), 8.07 (1H, d), 8.49 (1H, d).

Example B106 4-[4-(1-Isoquinolylmethyl)phenyl]-2-methyl-3-butyn-1-ol

[0636]

[0637] The title compound was obtained by treating the compound ofExample B105 in the same manner as in Example B47.

[0638]¹H-NMR (DMSO-d6) δ (ppm): 1.11 (3H, d), 2.60-2.70 (1H, m), 3.28(1H, d), 3.44 (1H, d), 4.58 (2H, s), 4.85-4.90 (1H, m), 7.23 (4H, s),7.61 (1H, dd), 7.70 (1H, d), 7.71 (1H, dd), 7.93 (1H, d), 8.25 (1H, d),8.42 (1H, d).

Example B107 1-([1-(t-Butyl)-1,1-dimethylsilyl]oxy}-3-butyn-2-ol

[0639]

[0640] Ethynyl magnesium bromide in tetrahydrofuran (0.5 M, 90 ml) wasadded to anhydrous tetrahydrofuran (20 ml) cooled to −78° C. undernitrogen atmosphere. A solution of t-butyldimethylsiloxyacetaldehyde(6000 mg) in tetrahydrofuran (30 ml) was added dropwise, and theeresulting mixture was stirred at −78° C. for 45 minutes, warmed to roomtemperature, stirred for 1 hour 40 minutes, then cooled on ice. After asaturated aqueous ammonium chloride solution was added, the reactionmixture was extracted with ether, washed with water and saturated brine,dried over anhydrous magnesium sulfate, and then filtered through silicagel. The filtrate was concentrated under reduced pressure to give thetitle compound (8.55 g). This compound was used in the followingreaction without further purification.

[0641]¹H-NMR (CDCl₃) δ (ppm): 0.08 (6H, s), 0.91 (9H, s), 2.43 (1H, d),2.60-2.66 (1H, m), 3.65-3.70 (1H, m), 3.73-3.81 (1H, m), 4.38-4.42 (1H,m).

Example B108 1-{[1-(t-Butyl)-1,1-dimethylsilyl]oxy}methyl)-2-propynylAcetate

[0642]

[0643] The title compound was obtained by treating the compound ofExample B107 in the same manner as in Example B38.

[0644]¹H-NMR (CDCl₃) δ (ppm): 0.08 (6H, s), 0.90 (9H, s), 2.11 (3H, s),2.44 (1H, d), 3.80-3.88 (2H, m), 5.41-5.55 (1H, m).

Example B109 4-[4-(1-Isoquinolylmethyl)phenyl]-3-butyn-1,2-diol

[0645]

[0646] The compound of Example B41 and the compound of Example B108 weretreated in the same manner as in Example B42 to give the couplingproduct. The title compound was obtained by deprotecting the hydroxylprotecting group of the coupling product in the same manner as inExample B47.

[0647]¹H-NMR (DMSO-d6) δ (ppm): 3.40-3.45 (1H, m), 3.70-3.82 (1H, m),4.30-4.35 (1H, m) 4.63 (2H, s) 4.90 (1H, t), 5.46 (1H, d), 7.25-7;30(4H, m), 7.62 (1H, dd), 7.71 (1H, d), 7.73 (1H, dd), 7.94 (1H, d), 8.28(1H, d), 8.43 (1H, d).

Example B1101-{4-[2-(2,2-Dimethyl-1,3-dioxolan-4-yl)-1-ethynyl]benzyl}isoquinoline

[0648]

[0649] 2,2-Dimethoxypropane (0.36 ml), 10-camphorsulfonic acid (43 mg),and molecular sieves (4 Å) were added to a solution of the compound ofExample B109 (34 mg) in dimethylformamide (2 ml), and this reactionmixture was stirred at 75° C. for 9 hours. After an saturated aqueoussodium carbonate solution was added, the reaction mixture was extractedwith ethyl acetate, washed with water, dried over anhydrous magnesiumsulfate, and then concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compound(14 mg).

[0650]¹H-NMR (CDCl₃) δ (ppm): 1.40 (3H, s), 1.50 (3H, s), 3.97 (1H, dd),4.21 (1H, dd), 4.66 (2H, s), 4.91 (1H, dd), 7.19 (2H, d), 7.32 (2H, d),7.52 (1H, dd), 7.65-7.78 (2H, m), 8.08 (1H, d), 8.09 (1H, d), 8.49 (1H,d).

Example B111 t-Butyl{[2-(1-ethoxyethoxy)-3-butynyl]oxy}dimethylsilane

[0651]

[0652] Ethyl vinyl ether (1.21 ml) and pyridinium p-toluenesulfonate(317 mg) were added to a solution of1-{[1-(t-butyl)-1,1-dimethylsilyl]oxy}-3-butyn-2-ol (1687 mg) inmethylene chloride (90 ml), and this mixture was stirred at roomtemperature for 1 hour. The methylene chloride layer was washed with asaturated aqueous sodium hydrogencarbonate solution and saturated brine,dried over anhydrous magnesium sulfate, then concentrated under reducedpressure to give the title compound (1962 mg). This compound was used inthe following reaction without further purification.

[0653]¹H-NMR (DMSO-d6) δ (ppm): 0.00 (6H, s), 0.81 (9H, s), 1.01-1.07(3H, m), 1.10-1.20 (1H, m), 1.18 (3H, d), 3.35-3.63 (4H, m), 4.18-4.27(1H, m), 4.74 (0.5H, q), 4.81 (0.5H, q).

Example B1121-{4-[4-{[1-(t-Butyl)-1,1-dimethylsilyl]oxy}-3-(1-ethoxyethoxy)-1-butynyl]benzyl}isoquinoline

[0654]

[0655] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B111 in the same manner as inExample B42.

[0656]¹H-NMR (DMSO-d6) δ (ppm): 0.00 (6H, s), 0.80 (9H, s), 1.01-1.05(3H, m), 1.19 (3H, d), 3.39-3.70 (4H, m), 4.41 (0.5H, t), 4.48 (0.5H,t), 4.59 (2H, s), 4.79 (0.5H, q), 4.87 (0.5H, q), 7.20-7.30 (4H, m),7.58 (1H, dd), 7.68 (1H, d), 7.69 (1H, dd), 7.91 (1H, d), 8.24 (1H, d),8.38 (1H, d).

Example B1131-{[1-(t-Butyl)-1,1-dimethylsilyl]oxy}4-[4-(1-isoquinolylmethyl)phenyl]-3-butyn-2-ol

[0657]

[0658] Pyridinium p-toluenesulfonate (486 mg) was added to a solution ofthe compound of Example B112 (474 mg) in methanol (15 ml), and thisreaction mixture was stirred at room temperature for 24 hours. Afterethyl acetate was added, the reaction mixture was washed with asaturated aqueous sodium hydrogencarbonate solution and saturated brine,dried over anhydrous magnesium sulfate, and then concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (265 mg).

[0659]¹H-NMR (DMSO-d6) δ (ppm): 0.01 (6H, s), 0.82 (9H, s), 3.55-3.62(2H, m), 4.30-4.39 (1H, m), 4.61 (2H, s), 5.51 (1H, d), 7.20-7.27 (4H,m), 7.50-7.63 (1H, m), 7.67-7.74 (2H, m), 7.92 (1H, d), 8.27 (1H, d),8.41 (1H, d).

Example B1141-(t-Butyl)-1,1-dimethylsilyl{2-fluoro-4-[4-(1-isoquinolylmethyl)phenyl]-3-butynyl}Ether

[0660]

[0661] A solution of the compound of Example B113 (116 mg) in methylenechloride (2 ml) was added dropwise to a solution of (diethylamino)sulfurtrifluoride (44 μl) in methylene chloride (2 ml) cooled to −78° C. undernitrogen atmosphere. Upon stirring for 15 minutes, the reaction mixturewas stirred at room temperature for another 8 hours. A saturated aqueoussodium hydrogencarbonate solution was added, the resulting reactionmixture was extracted with methylene chloride. The methylene chloridelayer was washed with water, dried over anhydrous magnesium sulfate, andthen concentrated under reduced pressure. The residue was purified bysilica gel column chromatography to give the title compound (42 mg).

[0662]¹H-NMR (CDCl₃) δ (ppm): 0.10 (6H, s), 0.91 (9H, s), 3.83-4.00 (2H,m), 4.67 (2H, s), 5.17 (1H, ddd), 7.22 (2H, d), 7.34 (2H, d), 7.53 (1H,dd), 7.58 (1H, d), 7.65 (1H, dd), 7.83 (1H, d), 8.08 (1H, d), 8.50 (1H,d).

Example B115 2-Fluoro-4-[4-(1-isoquinolylmethyl)phenyl]-3-butyn-1-ol

[0663]

[0664] The title compound was obtained by treating the compound ofExample B114 in the same manner as in Example B47.

[0665]¹H-NMR (CDCl₃) δ (ppm): 1.31 (1H, brs), 3.77-3.95 (2H, m), 4.67(2H, s), 5.35 (1H, ddd), 7.22 (2H, d), 7.35 (2H, d), 7.53 (1H, dd), 7.58(1H, d), 7.65 (1H, dd), 7.83 (1H, d), 8.07 (1H, d), 8.50 (1H, d).

Example B1161-(t-Butyl)-1,1-dimethylsilyl{6-[4-(1-isoquinolylmethyl)-phenyl]-5-hexynyl}Ether

[0666]

[0667] The title compound was obtained by treating the compound ofExample B41 and t-butyl(5-hexynyloxy)dimethylsilane in the same manneras in Example B42.

[0668]¹H-NMR (CDCl₃) δ (ppm): 0.04 (6H, s), 0.88 (9H, s), 1.55-1.70 (4H,m), 2.39 (2H, t), 3.64 (2H, t), 4.65 (2H, s), 7.17 (2H, d), 7.27 (2H,d), 7.51 (1H, dd), 7.55 (1H, d), 7.64 (1H, dd), 7.82 (1H, d), 8.08 (1H,d), 8.49 (1H, d):

Example B117 6-[4-(1-Isoquinolylmethyl)phenyl]-5-hexyn-1-ol

[0669]

[0670] The title compound was obtained by treating the compound ofExample B116 in the same manner as in Example B47.

[0671]¹H-NMR (CDCl₃) δ (ppm): 1.60-1.80 (4H, m), 2.42 (2H, t), 3.69 (2H,t), 4.65 (2H, s), 7.17 (2H, d), 7.27 (2H, d), 7.52 (1H, dd), 7.57 (1H,d), 7.64 (1H, dd), 7.81 (1H, d), 8.08 (1H, d), 8.49 (1H, d).

[0672] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B118 6-[4-(1-Isoquinolylmethyl)phenyl]-1-hexanol

[0673]

[0674] Treating the compound of Example B117 in the same manner as inExample B43, the obtained residue was separated and purified by LC-MS[eluent: an acetonitrile solution containing 0.1% trifluoroacetic acid:an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)] to give the title compound.

[0675] MS m/z(ESI:MH⁺): 320.2.

Example B119 2-(4-Pentynyloxy)tetrahydro-2H-pyran

[0676]

[0677] The title compound was obtained by treating 4-pentyn-1-ol in thesame manner as in Example B91.

[0678]¹H-NMR (CDCl₃) δ (ppm): 1.50-1.90 (8H, m), 1.95 (1H, t), 2.30-2.35(2H, m), 3.46-3.54 (2H, m), 3.80-3.90 (2H, m), 4.60 (1H, dd).

Example B1201-{4-[5-(Tetrahydro-2H-2-pyranyloxy)-1-pentynyl]benzyl}isoquinoline

[0679]

[0680] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B119 in the same manner as inExample B42.

[0681]¹H-NMR (CDCl₃) δ (ppm): 1.49-1.90 (8H, m), 2.49 (2H, t), 3.47-3.54(2H, m), 3.82-3.90 (2H, m), 4.60 (1H, dd), 4.65 (2H, s), 7.17 (2H, d),7.27 (2H, d), 7.52 (1H, dd), 7.58 (1H, d), 7.64 (1H, dd), 7.82 (1H, d),8.09 (1H, d), 8.49 (1H, d).

Example B121 5-[4-(1-Isoquinolylmethyl)phenyl]-4-pentyn-1-ol

[0682]

[0683] The title compound was obtained by treating the compound ofExample B120 in the same manner as in Example B47.

[0684]¹H-NMR (CDCl₃) δ (ppm): 1.80-1.88 (2H, m), 2.51 (2H, t), 3.80 (2H,t), 4.65 (2H, s), 7.18 (2H, d), 7.29 (2H, d), 7.52 (1H, dd), 7.58 (1H,d), 7.65 (1H, dd), 7.82 (1H, d), 8.09 (1H, d), 8.49 (1H, d).

[0685] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B122 5-[4-(1-Isoquinolylmethyl)phenyl]-4-pentynylcyanide

[0686]

[0687] The title compound was obtained by treating the compound ofExample B41 and 5-cyano-1-pentyne in the same manner as in Example B42.

[0688]¹H-NMR (CDCl₃) δ (ppm): 1.85-1.98 (2H, m), 2.40-2.60 (4H, m), 4.66(2H, s), 7.20 (2H, d), 7.28 (2H, d), 7.53 (1H, dd), 7.58 (1H, d), 7.65(1H, dd), 7.83 (1H, d), 8.09 (1H, d), 8.50 (1H, d).

Example B123 1-[4-(3-Methyl-1-butynyl)benzyl]isoquinoline

[0689]

[0690] The title compound was obtained by treating the compound ofExample B41 and 3-methyl-1-butyne in the same manner as in Example B42.

[0691]¹H-NMR (CDCl₃) δ (ppm): 1.23 (6H, d), 2.70-2.78 (1H, m), 4.65 (2H,s), 7.18 (2H, d), 7.28 (2H, d), 7.51 (1H, dd), 7.58 (1H, d), 7.64 (1H,dd), 7.82 (1H, d), 8.08 (1H, d), 8.50 (1H, d).

Example B124 1-[4-(5-Methyl-1-hexynyl)benzyl]isoquinoline

[0692]

[0693] The title compound was obtained by treating the compound ofExample B41 and 5-methyl-1-hexyne in the same manner as in Example B42.

[0694]¹H-NMR (CDCl₃) δ (ppm): 0.91 (6H, d), 1.47 (2H, dt), 1.68-1.77(1H, m), 2.37 (2H, t), 4.65 (2H, s), 7.17 (2H, d), 7.28 (2H, d), 7.52(1H, dd), 7.57 (1H, d), 7.64 (1H, dd), 7.81 (1H, d), 8.09 (1H, d), 8.49(1H, d).

Example B125 4-Pentynamide

[0695]

[0696] 1-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (6775 mg) andammonium hydrogencarbonate (5905 mg) were added to a solution of4-pentynoic acid (2446 mg) in chloroform (75 ml), and this reactionmixture was stirred at room temperature for 17.5 hours. The reactionmixture was filtered through celite and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (249 mg).

[0697]¹H-NMR (DMSO-d6) δ (ppm): 2.21 (2H, t), 2.29-2.33 (2H, m), 2.73(1H, t), 6.78-6.88 (1H, m), 7.28-7.38 (1H, m).

Example B126 5-[4-(1-Isoquinolylmethyl)phenyl]-4-pentynamide

[0698]

[0699] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B125 in the same manner as inExample B42.

[0700]¹H-NMR (DMSO-d6) δ (ppm): 2.51 (2H, t), 2.85 (2H, t), 3.70 (2H,brs), 4.59 (2H, s), 7.05 (2H, d), 7.23 (2H, d), 7.61 (1H, dd), 7.70 (1H,d), 7.72 (1H, dd), 7.94 (1H, d), 8.30 (1H, d), 8.43 (1H, d).

Example B127 t-Butyl 4-Pentynoate

[0701]

[0702] Benzyltriethylammonium chloride (5.92 g), potassium carbonate(93.4 g), and t-butyl bromide (143 ml) were added to a solution of4-pentynoic acid (2550 mg) in N,N-dimethylacetamide (230 ml), and thisreaction mixture was stirred at 55° C. for 24 hours. After water wasadded, the reaction mixture was extracted with ethyl acetate, washedwith water, dried over anhydrous magnesium chloride, and then filteredthrough silica gel. The filtrate was concentrated under reduced pressureto give the title compound (2.10 g) This compound was used in thefollowing reaction without further purification.

[0703]¹H-NMR (CDCl₃) δ (ppm): 1.46 (9H, s), 1.96-1.97 (1H, m), 2.45-2.47(4H, m).

Example B128 t-Butyl 5-[4-(1-Isoquinolylmethyl)phenyl]-4-pentynoate

[0704]

[0705] The title compound was obtained by treating the compound ofExample B41 and the compound of Example B127 in the same manner as inExample B42.

[0706]¹H-NMR (CDCl₃) δ (ppm): 1.45 (9H, s), 2.49 (2H, t), 2.64 (2H, t),4.64 (2H, s), 7.21 (2H, d), 7.26 (2H, d), 7.52 (1H, dd), 7.57 (1H, d),7.64 (1H, dd), 7.82 (1H, d), 8.09 (1H, d), 8.49 (1H, d).

Example B129 5-[4-(1-Isoquinolylmethyl)phenyl]-4-pentynoic Acid

[0707]

[0708] Treating the compound of Example B128 in the same manner as inExample B69, the obtained residue was separated and purified by LC-MS[eluent: an acetonitrile solution containing 0.1% trifluoroacetic acid:an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)] to give the title compound.

[0709] MS m/z(ESI:MH⁺): 316.1.

[0710] The following compounds were synthesized as follows. That is, thetitle compound was obtained by reacting the compound of Example B41 withvarious reactants described below, according to Example B33. The variousreactants are acrylamide, N,N-dimethylacrylamide, t-butyl acrylate, andmethyl vinyl sulfone. Furthermore, the coupling product obtained in thismanner was subjected to either the reduction according to Example B39 orthe deprotection of t-butyl ester according to Example B40, or both. Theresulting product was purified by silica gel column chromatography or byLC-MS [eluent: an acetonitrile solution containing 0.1% trifluoroaceticacid: an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)].

Example B130 (E)-3-[4-(1-Isoquinolylmethyl)phenyl]-2-propenamide

[0711]

[0712] MS m/z(ESI:MH⁺): 289.3.

Example B131 3-[4-(1-Isoquinolylmethyl)phenyl]-2-propanamide

[0713]

[0714] MS m/z(ESI:MH⁺): 291.2.

Example B132N,N-Dimethyl-(E)-3-[4-(1-isoquinolylmethyl)phenyl]-2-propenamide

[0715]

[0716] MS m/z(ESI:MH⁺): 317.3.

Example B133 N,N-Dimethyl-3-[4-(1-isoquinolylmethyl)phenyl]propanamide

[0717]

[0718] MS m/z(ESI:MH⁺): 319.1.

Example B134 t-Butyl (E)-3-[4-(1-isoquinolylmethyl)phenyl]-2-propenoate

[0719]

[0720]¹H-NMR (CDCl₃) δ (ppm): 1.51 (9H, s), 4.68 (2H, s), 6.28 (1H, d),7.27 (2H, d), 7.39 (2H, d), 7.49-7.60 (3H, m), 7.65 (1H, dd), 7.82 (1H,d), 8.11 (1H, d), 8.50 (1H, d).

Example B135 (E)-3-[4-(1-Isoquinolylmethyl)phenyl]-2-propenoic Acid

[0721]

[0722] MS m/z(ESI:MH⁺): 290.2.

Example B136 t-Butyl 3-[4-(1-Isoquinolylmethyl)phenyl]propanoate

[0723]

[0724]¹H-NMR (CDCl₃) δ (ppm): 1.37 (9H, s), 2.47 (2H, t), 2.83 (2H, t),4.64 (2H, s), 7.07 (2H, d), 7.19 (2H, d), 7.52 (1H, dd), 7.56 (1H, d),7.63 (1H, dd), 7.81 (1H, d), 8.14 (1H, d), 8.49 (1H, d).

Example B137 3-[4-(1-Isoquinolylmethyl)phenyl]propanoic Acid

[0725]

[0726] MS m/z(ESI:MH⁺): 292.1.

Example B138 (E)-2-[4-(1-Isoquinolylmethyl)phenyl]-1-ethenylMethylsulfone

[0727]

[0728] MS m/z(ESI:MH⁺): 324.1.

Example B139 1-{4-[2-(Methylsulfonyl)ethyl]benzyl}isoquinoline

[0729]

[0730] MS m/z(ESI:MH⁺): 326.1.

Example B1402-Benzoyl-6,7-dimethoxy-1,2-dihydro-1-isoquinolinecarbonitrile

[0731]

[0732] An aqueous potassium cyanide (1.0 g, 16 mmol) solution (2.3 ml)and benzoyl chloride (1.1 ml, 9.5 mmol) were added to a solution of6,7-dimethoxyisoquinoline (1.0 g, 5.3 mmol), which was synthesizedaccording to Tetrahedron, 37 (23), 3977 (1981), in methylene chloride(6.0 ml), and this reaction mixture was stirred while heating underreflux for 2 hours. The reaction mixture was cooled to room temperature,filtered through celite, and washed with methylene chloride and water.After the obtained filtrate was separated, the methylene chloride layerwas washed successively with water, 2 N hydrochloric acid, water, and 2N sodium hydroxide, dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound (573 mg).

[0733]¹H-NMR (CDCl₃) δ (ppm): 3.92 (3H, s), 3.94 (3H, s), 5.99 (1H, d),6.51-6.55 (2H, m), 6.73 (1H, s), 6.85 (1H, s), 7.45-7.49 (2H, m),7.53-7.56 (1H, m), 7.58-7.61 (2H, m).

Example B141 1-(4-Butylbenzyl)-6,7-dimethoxyisoquinoline

[0734]

[0735] The title compound was obtained by treating the compound ofExample B140 and the compound of Example B1 in the same manner as inExample B2.

[0736]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.27-1.36 (2H, m), 1.51-1.58(2H, m), 2.54 (2H, t), 3.88 (3H, s), 4.01 (3H, s), 4.57 (2H, s), 7.05(1H, s), 7.07 (2H, d), 7.19 (2H, d), 7.32 (1H, s), 7.43 (1H, d), 8.37(1H, d).

Example B142 1-(3-Methoxyphenyl)-2-nitro-1-ethanol

[0737]

[0738] An aqueous sodium hydroxide solution (1.5 g of sodium hydroxide(37 mmol) was dissolved in 15 ml of water) was added dropwise to asolution of m-anisaldehyde (5.0 g, 37 mmol) and nitromethane (4.0 ml, 73mmol) in methanol (50 ml) keeping the temperature of the solution at nothigher than 30° C. The reaction mixture was then stirred at roomtemperature for 4 hours. Upon cooling on ice, an aqueous acetic acidsolution (glacial acetic acid (37 mmol) was dissolved in 250 ml ofwater) was added, the resulting reaction mixture was extracted withethyl acetate. The ethyl acetate layer was washed successively withwater and a 5% aqueous sodium hydrogencarbonate solution, dried overanhydrous magnesium sulfate, then concentrated under reduced pressure.The residue was purified by silica gel column chromatography to give thetitle compound (6.09 g).

[0739]¹H-NMR (CDCl₃) δ (ppm): 3.83 (3H, s), 4.52 (1H, dd), 4.61 (1H,dd), 4.76-4.78 (1H, m), 5.44-5.48 (1H, m), 6.90 (1H, dd), 6.96-6.98 (2H,m), 7.25-7.34 (1H, m).

Example B143 2-Amino-1-(3-methoxyphenyl)-1-ethanol

[0740]

[0741] Palladium-carbon (10%, 0.64 g) and ammonium formate (4.8 g) wereadded to a mixed solution of the compound of Example B142 (3.0 g, 15mmol) in tetrahydrofuran (43 ml) and methanol (43 ml), and this mixturewas stirred at room temperature for 18 hours. The catalyst was removedby filtration, the filtrate was diluted with ether, precipitates wereremoved by filtration, and the obtained filtrate was concentrated togive the title compound (1.82 g). This compound was used in thefollowing reaction without further purification.

Example B144 2-(4-Butylphenyl)acetic Acid

[0742]

[0743] Thionyl chloride (4.7 ml, 66 mmol) was added dropwise to asolution of 4-n-butylbenzyl alcohol (9.6 g, 59 mmol) in ether (120 ml),and this mixture was stirred at room temperature for 2 hours. Thesolvent was removed under reduced pressure, and excess thionyl chloridewas removed by azeotropic distillation with benzene. The residue wasdissolved in dimethyl sulfoxide (50 ml), sodium cyanide (86 g, 1.8 mol)and n-tetrabutylammonium iodide (2.2 g, 5.9 mmol) were added to thissolution, and the resulting mixture was stirred at room temperature for16 hours. Water was added, and this mixture was extracted with ethylacetate. The ethyl acetate layer was washed successively with water andsaturated brine, dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give n-butylphenylacetonitrile (8.2 g) as ayellow oil. Next, concentrated sulfuric acid (48 ml) was added dropwiseto water (58 ml), this solution was cooled to 50° C., andn-Butylphenylacetonitrile (8.2 g) obtained above was added dropwise tothe solution. The resulting mixture was stirred while heating underreflux for 16 hours. Upon cooling to room temperature, the precipitatedcrystals were collected by filtration, washed with water, and dissolvedin a 0.1 N aqueous sodium hydroxide solution (200 ml). Norit (5 g) wasadded, and this mixture was stirred and refluxed for 2 hours. AfterNorit was removed by filtration through celite, the filtrate was cooledto room temperature and acidified with 1 N hydrochloric acid toprecipitate crystals. The precipitated crystals were collected byfiltration, washed with water, and dried to give the title compound (3.5g).

[0744]¹H-NMR (CDCl₃) δ (ppm): 0.93 (3H, t), 1.30-1.40 (2H, m), 1.53-1.62(2H, m), 2.59 (2H, t), 3.62 (2H, s), 7.15 (2H, d), 7.20 (2H, d).

[0745] The OH of the carboxyl group was not observed in the NMRspectrum.

Example B145N-[2-Hydroxy-2-(3-methoxyphenyl)ethyl]-2-(4-butylphenyl)acetamide

[0746]

[0747] Thionyl chloride (0.76 ml, 10 mmol) was added to a solution ofthe compound of Example B144 (1.0 g, 5.2 mmol) in benzene (10 ml), andthe mixture was stirred under reflux for 2 hours. Upon concentration,excess thionyl chloride was removed by azeotropic distillation withbenzene. The obtained residue and the compound of Example B143 (0.87 g,5.2 mmol) were dissolved in ether (5 ml), an aqueous sodium hydroxidesolution (0.21 g of sodium hydroxide was dissolved in 4.2 ml of water)was added thereto, and the mixture was stirred vigorously at roomtemperature for 30 minutes. The ether layer was separated andconcentrated under reduced pressure to give the title compound (600 mg).

[0748]¹H-NMR (CDCl₃) δ (ppm): 0.94 (3H, t), 1.31-1.40 (2H, m), 1.57-1.63(2H, m), 2.60 (2H, m), 3.30-3.37 (1H, m), 3.56 (2H, s), 3.60-3.66 (1H,m), 3.80 (3H, s), 3.81 (1H, d), 4.79-4.81 (1H, m), 6.80-6.89 (3H, m),7.10 (2H, d), 7.16 (2H, d), 7.20-7.25 (1H, m).

Example B146 1-(4-Butylbenzyl)-6-methoxyisoquinoline

[0749]

[0750] Phosphorus oxychloride (1.6 ml) was added to a solution of thecompound of Example B145 (600 mg, 1.7 mmol) in acetonitrile (15 ml), andthe mixture was stirred under reflux for 1 hour 30 minutes. The mixturewas cooled on ice, made alkaline with a 5% aqueous sodiumhydrogencarbonate solution, extracted with ethyl acetate, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (82 mg).

[0751]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.58(2H, m), 2.53 (2H, t), 3.92 (3H, s), 4.57 (2H, s), 7.05-7.07 (3H, m),7.13-7.18 (3H, m), 7.45 (1H, d), 8.06 (1H, d), 8.41 (1H, d).

Example 147 1-(4-Butylbenzyl)-6-isoquinolinol

[0752]

[0753] A 47% hydrobromic acid solution was added to the compound ofExample B146 (82 mg), and the mixture was stirred under reflux for 19hours. The mixture was concentrated under reduced pressure, water wasadded, and the resulting mixture was neutralized with sodium carbonateto precipitate crystals. The obtained crystals were collected byfiltration, washed with water, and then dried to give the title compound(74 mg).

[0754]¹H-NMR (CD₃OD) δ (ppm): 0.89 (3H, t), 1.25-1.34 (2H, m), 1.49-1.57(2H, m), 2.52 (2H, t), 4.63 (2H, s), 7.03-7.13 (6H, m), 7.49 (1H, d),8.10 (1H, d), 8.18 (1H, d).

Example B148 1-(4-Butylbenzyl)-6-propoxyisoquinoline

[0755]

[0756] Silver carbonate (40 mg, 0.14 mmol) was added to a solution ofthe compound of Example B147 (20 mg, 0.069 mmol) and 1-iodopropane (0.4ml, 4.1 mmol) in toluene (1.0 ml), and the mixture was stirred in thedark at 50° C. for 4 hours. Upon cooling to room temperature, themixture was filtered through celite and washed with a mixed solution oftoluene and methanol (9:1). The obtained filtrate was concentrated underreduced pressure, and the resulting residue was purified by silica gelcolumn chromatography to give the title compound (13 mg).

[0757]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.08 (3H, t), 1.30-1.33 (2H,m), 1.51-1.57 (2H, m), 1.86-1.91 (2H, m), 2.54 (2H, t), 4.05 (2H, t),4.58 (2H, s), 7.05-7.07 (3H, m), 7.14-7.18 (3H, m), 7.43-7.44 (1H, m),8.05-8.07 (1H, m), 8.40-8.41 (1H, m).

Example B149 1-(4-Butylbenzyl)-6-(2-piperidinoethoxy)isoquinoline

[0758]

[0759] The title compound was obtained in the same manner as in Example148.

[0760]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.26-1.36 (2H, m), 1.46-1.57(8H, m), 2.50-2.54 (6H, m), 2.83-2.86 (2H, m), 4.23 (2H, t), 4.56 (2H,s), 7.04-7.06 (3H, m), 7.13-7.17 (3H, m), 7.43 (1H, d), 8.04 (1H, d),8.40 (1H, d).

Example B150N-({[1-(4-Butylbenzyl)-6-isoquinolyl]oxy}ethyl)-N,N-dimethylamine

[0761]

[0762] The title compound was obtained in the same manner as in Example148.

[0763]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.26-1.36(2H, m), 1.49-1.57(2H, m), 2.37 (6H, s), 2.52 (2H, t), 2.80 (2H, t), 4.19 (2H, t), 4.57(2H, s), 7.04-7.06 (3H, m), 7.15-7.19 (3H, m), 7.43 (1H, d), 8.05 (1H,d), 8.40 (1H, d).

Example B151 2-Benzoyl-7-methoxy-1,2-dihydro-1-isoquinolinecarbonitrile

[0764]

[0765] The title compound was obtained by treating7-methoxyisoquinoline, which was synthesized according to Tetrahedron,27, 1253 (1971), in the same manner as in Example B140.

[0766]¹H-NMR (CDCl₃) δ (ppm): 3.87 (3H, s), 6.03 (1H, brd), 6.56-6.54(2H, m), 6.90 (1H, s), 6.95 (1H, dd), 7.17 (1H, d), 7.46-7.50 (2H, m),7.54-7.62 (3H, m).

Example B152 1-(4-Butylbenzyl)-7-methoxyisoquinoline

[0767]

[0768] The title compound was obtained by treating the compound ofExample B1 and the compound of Example B151 in the same manner as inExample B2.

[0769]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.56-1.58(2H, m), 2.55 (2H, t), 3.82 (3H, s), 4.59 (2H, s), 7.07 (2H, d), 7.20(2H, d), 7.26-7.29 (1H, m), 7.35 (1H, d), 7.49 (1H, d), 7.70 (1H, d),8.38-8.40 (1H, m).

Example B153 1-(4-Bromobenzyl)-7-methoxyisoquinoline

[0770]

[0771] The title compound was obtained by treating the compound ofExample B31 and the compound of Example B151 in the same manner as inExample B2.

[0772]¹H-NMR (CDCl₃) δ (ppm): 3.84 (3H, s), 4.57 (2H, s), 7.14-7.16 (2H,m), 7.26 (1H, s), 7.29-7.32 (1H, m), 7.37-7.39 (2H, m), 7.51 (1H, d),7.73 (1H, d), 8.39 (1H, d).

Example B154 1-(4-Butylbenzyl)-7-isoquinolinol

[0773]

[0774] The title compound was obtained by treating the compound ofExample B152 in the same manner as in Example B147.

[0775]¹H-NMR (DMSO-d₆) δ (ppm): 0.83 (3H, t), 1.21-1.26 (2H, m),1.44-1.48 (2H, m), 4.68 (2H, s), 7.11 (2H, d), 7.18 (2H, d), 7.59-7.62(2H, m), 8.10-8.17 (2H, m), 8.38 (1H, d), 10.9 (1H, brs); (The twomethylene protons of the butyl group overlapped with the DMSO signal andcould not be observed.)

Example B155 1-(4-Butylbenzyl)-7-isoquinolyl Trifluoromethanesulfonate

[0776]

[0777] 4-Nitrophenol triflate (0.72 g, 2.7 mmol), which was synthesizedaccording to J. Org. Chem., 64,7638 (1999), and potassium carbonate (1.1g, 8.1 mmol) were added to a solution of the compound of Example B154(1.0 g, 2.7 mmol) in dimethylformamide (30 ml), and the mixture wasstirred at room temperature for 2 hours. After water was added, theresulting mixture was extracted with ethyl acetate. The ethyl acetatelayer was washed with 1 N sodium hydroxide and saturated brine, driedover magnesium sulfate, and then concentrated under reduced pressure.The residue was purified by silica gel column chromatography to give thetitle compound (1.0 g).

[0778]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.27-1.37 (2H, m), 1.51-1.59(2H, m), 2.54 (2H, t), 5.10 (2H, s), 6.38 (1H, s), 6.95 (2H, d), 7.04(2H, d), 7.44 (1H, d), 7.55 (1H, d), 7.75 (1H, d), 8.45 (1H, d).

Example B156 1-(4-Butylbenzyl)-7-isoquinolinecarbonitrile

[0779]

[0780] Zinc cyanide (215 mg, 1.8 mmol),tetrakis(triphenylphosphine)palladium (41 mg, 0.035 mmol), and lithiumchloride (120 mg, 2.8 mmol) were added to a solution of the compound ofExample B155 (400 mg, 0.95 mmol) in dimethylformamide (2 ml) undernitrogen atmosphere, and the mixture was stirred at 120° C. for 2 hours.After cooling to room temperature, saturated sodium hydrogencarbonatewas added, and the resulting mixture was extracted with ethyl acetate.The ethyl acetate layer was washed with saturated brine, dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (71 mg).

[0781]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.26-1.35 (2H, m), 1.47-1.55(2H, m), 2.50 (2H, t), 4.91 (2H, s), 6.97 (2H, d), 7.07 (2H, d),7.28-7.31 (1H, m), 7.42 (1H, d), 7.51 (1H, d), 7.74 (1H, d), 8.34 (1H,d).

Example B1571-(4-Butylbenzyl)-7-[2-(1,1,1-trimethylsilyl)-1-ethynyl]isoquinoline

[0782]

[0783] Palladium acetate (11 mg, 0.047 mmol),1,1′-bis(diphenylphosphino)ferrocene (72 mg, 0.13 mmol), and lithiumchloride (25 mg, 0.59 mmol) were added to a solution of the compound ofExample B155 (100 mg, 0.24 mmol) and trimethylsilylacetylene (65 μl,0.47 mmol) in dimethylformamide (3.0 ml), and the reaction system waspurged with nitrogen. Triethylamine (59 μl, 0.43 mmol) and copper iodide(2 mg, 0.018 mmol) were added, and the resulting mixture was stirred at80° C. for 21 hours, then cooled to room temperature. After water andethyl acetate were added for partition, the ethyl acetate layer waswashed with water, dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound (7.0 mg).

[0784]¹H-NMR (CDCl₃) δ (ppm): 0.28-0.32 (9H, m), 0.92 (3H, t), 1.32-1.38(2H, m), 1.54-1.57 (2H, m), 2.57 (2H, t), 4.63 (2H, s), 7.10 (2H, d),7.20 (2H, d), 7.52 (1H, d), 7.67-7.69 (1H, m), 7.75 (1H, d), 8.34 (1H,d), 8.51 (1H, d).

Example B158 1-(4-Butylbenzyl)-7-(1-ethynyl)isoquinoline

[0785]

[0786] Potassium carbonate (13 mg, 0.094 mmol) was added to a solutionof the compound of Example B157 (6 mg, 0.016 mmol) in methanol (1.0 ml),and the mixture was stirred at room temperature for 1 hour. Uponconcentration under reduced pressure, the obtained residue was purifiedby silica gel column chromatography to give the title compound (3.0 mg).

[0787]¹H-NMR (CDCl₃) δ (ppm): 0.91 (3H, t), 1.29-1.38 (2H, m), 1.52-1.57(2H, m), 2.55 (2H, t), 3.19 (1H, s), 4.62 (2H, s), 7.09 (2H, d), 7.20(2H, d), 7.53 (1H, d), 7.67-7.69 (1H, m), 7.77 (1H, d), 8.36 (1H, s),8.52 (1H, d).

Example B159 1-(4-Butylbenzyl)-7-ethylisoquinoline

[0788]

[0789] Palladium-carbon (10%, 5.0 mg) was added to a solution of thecompound of Example B158 (2.0 mg) in tetrahydrofuran (2.0 ml), and themixture was stirred at room temperature under nitrogen atmosphere (1atm) for 1 hour. The catalyst was removed by filtration, and thefiltrate was concentrated. The residue was purified by silica gel columnchromatography to give the title compound (0.21 mg).

[0790]¹H-NMR (CDCl₃) δ (ppm): 0.89 (6H, t), 1.25-1.32 (2H, m), 1.48-1.57(2H, m), 2.53 (2H, t), 2.80 (2H, q), 4.62 (2H, s), 7.06 (2H, d), 7.20(2H, d), 7.49-7.52 (2H, m), 7.73 (1H, d), 7.95 (1H, s), 8.43 (1H, d).

Example B1601-(4-Butylbenzyl)-7-[4-(tetrahydro-2H-2-pyranyloxy)-1-butynyl]-isoquinoline

[0791]

[0792] Palladium acetate (11 mg, 0.047 mmol), 1,1′-bis(diphenylphosphino) ferrocene (72 mg, 0.13 mmol), and lithium chloride(25 mg, 0.59 mmol) were added to a solution of the compound of ExampleB155 (100 mg, 0.24 mmol) and 2-(3-butynyloxy)tetrahydro-2H-pyran (73 mg,0.47 mmol) in dimethylformamide (3.0 ml), and the system was purged withnitrogen. Furthermore, triethylamine (59 μl, 0.43 mmol) and copperiodide (2 mg, 0.018 mmol) were added, and the resulting mixture wasstirred at 80° C. for 24 hours. The mixture was cooled to roomtemperature, water was added, and the resulting mixture was extractedwith ethyl acetate. The ethyl acetate layer was washed with water, driedover anhydrous magnesium sulfate, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (25 mg).

[0793]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.28-1.38 (2H, m), 1.52-1.67(6H, m), 1.72-1.79 (1H, m), 1.79-1.88 (1H, m), 2.54 (2H, t), 2.78 (2H,t), 3.53-3.56 (1H, m), 3.66-3.72 (1H, m), 3.91-3.99 (2H, m), 4.60 (2H,s), 4.71-4.73 (1H, m) 7.08 (2H, d), 7.19 (2H, d), 7.50 (1H, d),7.59-7.62 (1H, m), 7.72 (1H, d), 8.24 (1H, s), 8.48 (1H, d).

Example B161 4-[1-(4-Butylbenzyl)-7-isoquinolyl]-3-butyn-1-ol

[0794]

[0795] The title compound was obtained by treating the compound ofExample B160 in the same manner as in Example B29.

[0796]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.39 (2H, m), 1.51-1.57(2H, m), 1.83 (1H, brs), 2.55 (2H, t), 2.75 (2H, t), 3.84-3.89 (2H, m),4.60 (2H, s), 7.08 (2H, d), 7.18 (2H, d), 7.50 (1H, d), 7.60-7.62 (1H,m), 7.73 (1H, d), 8.25 (1H, s), 8.48 (1H, d).

Example B162 4-[1-(4-Butylbenzyl)-7-isoquinolyl]-1-butanol

[0797]

[0798] The title compound was obtained by treating the compound ofExample B161 in the same manner as in Example B30.

[0799]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.28-1.36 (2H, m), 1.50-1.59(4H, m), 1.67-1.77 (3H, m), 2.53 (2H, t), 2.79 (2H, t), 3.63 (2H, t),4.62 (2H, s), 7.06 (2H, d), 7.18 (2H, d), 7.47-7.52 (2H, m), 7.73 (1H,d), 7.92 (1H, s), 8.43 (1H, d).

Example B163 1-(4-Butylbenzyl)-7-propoxyisoquinoline

[0800]

[0801] The title compound was obtained by treating the compound ofExample B154 in the same manner as in Example B148.

[0802]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.05 (3H, t), 1.27-1.36 (2H,m), 1.50-1.56 (2H, m), 1.76-1.84 (2H, m), 2.53 (2H, t), 3.92 (2H, t),4.58 (2H, s), 7.06 (2H, d), 7.19 (2H, d), 7.26-7.29 (1H, m), 7.34 (1H,d), 7.48 (1H, d), 7.70 (1H, d), 8.38 (1H, d).

Example B164 1-(4-Butylbenzyl)-7-(2-piperidinoethoxy)isoquinoline

[0803]

[0804] The title compound was obtained in the same manner as in ExampleB148.

[0805]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.43-1.58(4H, m), 1.61-1.69 (4H, m), 2.51-2.55 (6H, m), 2.79 (2H, t), 4.11 (2H,t), 4.57 (2H, s), 7.06 (2H, d), 7.18 (2H, d), 7.28-7.30 (1H, m), 7.36(1H, d), 7.48 (1H, d), 7.70 (1H, d), 8.38 (1H, d).

Example B165N-(2-{[1-(4-Butylbenzyl)-7-isoquinolyl]oxy}ethyl)-N,N-dimethylamine

[0806]

[0807] The title compound was obtained in the same manner as in ExampleB148.

[0808]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.57(2H, m), 2.35 (6H, s), 2.53 (2H, t), 2.75 (2H, t), 4.06 (2H, t), 4.58(2H, s), 7.06 (2H, d), 7.18 (2H, d), 7.30-7.33 (1H, m), 7.36 (1H, d),7.48 (1H, d), 7.70 (1H, d), 8.39 (1H, d).

Example B166 1-(4-Butylbenzyl)-7-isoquinolyl-(2-morpholinoethyl)ether

[0809]

[0810] The title compound was obtained in the same manner as in ExampleB148.

[0811]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.58(2H, m), 2.51-2.58 (6H, m), 2.81 (2H, t), 3.75 (4H, t), 4.11 (2H, t),4.58 (2H, s), 7.06 (2H, d), 7.17 (2H, d), 7.28-7.31 (1H, m), 7.35 (1H,d), 7.49 (1H, d), 7.71 (1H, d), 8.39 (1H, d).

Example B167 7-(Benzyloxy)-1-(4-butylbenzyl)isoquinoline

[0812]

[0813] The title compound was obtained in the same manner as in ExampleB148.

[0814]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.54(2H, m), 2.54 (2H, t), 4.54 (2H, s), 5.06 (2H, s), 7.05 (2H, d), 7.14(2H, d), 7.34-7.43 (7H, m), 7.49 (1H, d), 7.72 (1H, d), 8.39 (1H, d).

Example B168 1-(4-Butylbenzyl)-7-(2-pyridylmethoxy)isoquinoline

[0815]

[0816] The title compound was obtained in the same manner as in ExampleB148.

[0817]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.49-1.57(2H, m), 2.52 (2H, t), 4.51 (2H, s), 5.25 (2H, s), 7.02 (2H, d), 7.14(2H, d), 7.24-7.27 (1H, m), 7.40 (1H, dd), 7.47-7.50 (3H, m), 7.68-7.72(1H, d), 7.74 (1H, d), 8.39 (1H, d), 8.64-8.66 (1H, m).

Example B169 1-(4-Butylbenzyl)-7-(3-pyridylmethoxy)isoquinoline

[0818]

[0819] The title compound was obtained in the same manner as in ExampleB148.

[0820]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.58(2H, m), 2.54 (2H, t), 4.57 (2H, s), 5.06 (2H, s), 7.07 (2H, d), 7.15(2H, d), 7.31-7.36 (2H, m), 7.42 (1H, d), 7.51 (1H, d), 7.74-7.76 (2H,m), 8.42 (1H, d), 8.61-8.62 (1H, m), 8.69-8.70 (1H, m).

Example B170 1-(4-Butylbenzyl)-7-(4-pyridylmethoxy)isoquinoline

[0821]

[0822] The title compound was obtained in the same manner as in ExampleB148.

[0823]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.56(2H, m), 2.54 (2H, t), 4.53 (2H, s), 5.09 (2H, s), 7.04 (2H, d), 7.09(2H, d), 7.33-7.39 (4H, m), 7.51 (1H, d), 7.76 (1H, d), 8.41 (1H, d),8.63-8.64 (2H, m).

Example B171 1-(4-Butylbenzyl)-7-[(2-methoxybenzyl)oxy]isoquinoline

[0824]

[0825] The title compound was obtained in the same manner as in ExampleB148.

[0826]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.57(2H, m), 2.53 (2H, t), 3.82 (3H, s), 4.52 (2H, s), 5.04 (2H, s),6.88-6.91 (1H, m), 6.99-7.02 (2H, m), 7.05 (2H, d), 7.14 (2H, d), 7.32(1H, t), 7.36 (1H, dd), 7.43 (1H, d), 7.48 (1H, d), 7.72 (1H, d), 8.39(1H, d).

Example B172 1-(4-Butylbenzyl)-7-[(3-methoxybenzyl)oxy]isoquinoline

[0827]

[0828] The title compound was obtained in the same manner as in ExampleB148.

[0829]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.56(2H, m), 2.53 (2H, t), 3.90 (3H, s), 4.53 (2H, s), 5.16 (2H, s),6.93-6.98 (2H, m), 7.03 (2H, d), 7.15 (2H, d), 7.30-7.35 (1H, m), 7.37(1H, dd), 7.41-7.43 (1H, m), 7.47 (1H, d), 7.51 (1H, d), 7.71 (1H, d),8.37 (1H, d).

Example B173 1-(4-Butylbenzyl)-7-[(4-methoxybenzyl)oxy]isoquinoline

[0830]

[0831] The title compound was obtained in the same manner as in ExampleB148.

[0832]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.37 (2H, m), 1.51-1.57(2H, m), 2.54 (2H, t), 3.83 (3H, s), 4.55 (2H, s), 4.99 (2H, s), 6.93(2H, d), 7.06 (2H, d), 7.15 (2H, d), 7.32-7.36 (3H, m), 7.44 (1H, d),7.48 (1H, d), 7.71 (1H, d), 8.38 (1H, d).

Example B1747-(1,3-Benzodioxol-5-ylmethoxy)-1-(4-butylbenzyl)isoquinoline

[0833]

[0834] The title compound was obtained in the same manner as in ExampleB148.

[0835]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.37 (2H, m), 1.51-1.57(2H, m), 2.54 (2H, t), 4.55 (2H, s), 4.95 (2H, s), 5.98 (2H, s), 6.82(1H, d), 6.88 (1H, dd), 6.92 (1H, d), 7.06 (2H, d), 7.15 (2H, d), 7.33(1H, dd), 7.42 (1H, d), 7.48 (1H, d), 7.72 (1H, d), 8.39 (1H, d).

Example B175 1-(4-Butylbenzyl)-7-[(2-nitrobenzyl)oxy]isoquinoline

[0836]

[0837] The title compound was obtained in the same manner as in ExampleB148.

[0838]¹H-NMR (CDCl₃) δ (ppm): 0.87 (3H, t), 1.26-1.34 (2H, m), 1.48-1.56(2H, m), 2.51 (2H, t), 4.53 (2H, s), 5.49 (2H, s), 7.03 (2H, d), 7.14(2H, d), 7.40 (1H, dd), 7.430-7.434 (1H, m), 7.45-7.49 (1H, m), 7.51(1H, d), 7.64-7.68 (1H, m), 7.76 (1H, d), 7.85-7.87 (1H, m), 8.22-8.24(1H, d), 8.41 (1H, d).

Example B176 1-(4-Butylbenzyl)-7-[(3-nitrobenzyl)oxy]isoquinoline

[0839]

[0840] The title compound was obtained in the same manner as in ExampleB148.

[0841]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.56(2H, m), 2.54 (2H, t), 4.55 (2H, s), 5.14 (2H, s), 7.05 (2H, d), 7.11(2H, d), 7.37-7.40 (2H, m), 7.51 (1H, d), 7.55-7.59 (1H, m), 7.73-7.78(2H, m), 8.19-8.22 (1H, m), 8.32-8.33 (1H, m), 8.42 (1H, d).

Example B177 1-(4-Butylbenzyl)-7-(phenethyloxy)isoquinoline

[0842]

[0843] The title compound was obtained in the same manner as in ExampleB148.

[0844]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.26-1.36 (2H, m), 1.49-1.57(2H, m), 2.52 (2H, t), 3.10 (2H, t), 4.18 (2H, t), 4.56 (2H, s), 7.04(2H, d), 7.16 (2H, d), 7.26-7.28 (4H, m), 7.33-7.35 (3H, m), 7.48 (1H,d), 7.70 (1H, d), 8.38-8.39 (1H, m).

Example B178 1-(4-Butylbenzyl)-7-(3-phenylpropoxy)isoquinoline

[0845]

[0846] The title compound was obtained in the same manner as in ExampleB148.

[0847]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.49-1.57(2H, m), 2.09-2.15 (2H, m), 2.52 (2H, t), 2.82 (2H, t), 3.97 (2H, t),4.55 (2H, s), 7.04 (2H, d), 7.16 (2H, d), 7.20-7.23 (3H, m), 7.27-7.33(4H, m), 7.48 (1H, d), 7.70 (1H, d), 8.38 (1H, d).

Example B179 1-(4-Butylbenzyl)-7-(2-cyclohexylethoxy)isoquinoline

[0848]

[0849] The title compound was obtained in the same manner as in ExampleB148.

[0850]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 0.94-1.02 (2H, m), 1.17-1.36(4H, m), 1.36-1.57 (4H, m), 1.65-1.76 (7H, m), 2.53 (2H, t), 3.98 (2H,t), 4.58 (2H, s), 7.06 (2H, d), 7.19 (2H, d), 7.25-7.28 (1H, m), 7.33(1H, d), 7.47 (1H, d), 7.69 (1H, d), 8.37 (1H, d).

Example B1806-Benzoyl-5,6-dihydro[1,3]dioxolo[4,5-g]isoquinoline-5-carbonitrile

[0851]

[0852] The title compound was obtained by treating[1,3]dioxolo[4,5-g]isoquinoline in the same manner as in Example B140.

[0853]¹H-NMR (CDCl₃) δ (ppm): 5.94-5.96 (1H, m), 6.03 (1H, d), 6.04 (1H,d), 6.47-6.54 (2H, m), 6.70 (1H, s), 6.83 (1H, s), 7.45-7.49 (2H, m),7.54-7.62 (3H, m).

Example B181 5-(4-Butylbenzyl)[1,3]dioxolo[4,5-g]isoquinoline

[0854]

[0855] The title compound was obtained by treating the compound ofExample B180 and the compound of Example B1 in the same manner as inExample B2.

[0856]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.28-1.37 (2H, m), 1.51-1.57(2H, m), 2.54 (2H, t), 4.50 (2H, s), 6.05 (2H, s), 7.05-7.07 (3H, m),7.16 (2H, d), 7.38 (7.40 (2H, m), 8.35 (1H, d).

Example B182 2-Benzoyl-6-bromo-1,2-dihydro-1-isoquinolinecarbonitrile

[0857]

[0858] The title compound was obtained by treating 6-bromoisoquinoline,which was synthesized according to J. Am. Chem. Soc., 183 (1942), in thesame manner as in Example B140.

[0859]¹H-NMR (CDCl₃) δ (ppm): 6.01 (1H, d), 6.53 (1H, brs), 6.70 (1H,brd), 7.24 (1H, d), 7.33 (1H, d), 7.47-7.51 (3H, m), 7.56 (3H, m).

Example B183 6-Bromo-1-(4-butylbenzyl)isoquinoline

[0860]

[0861] The title compound was obtained by treating the compound ofExample B182 and the compound of Example B1 in the same manner as inExample B2.

[0862]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.27-1.36 (2H, m), 1.50-1.58(2H, m), 2.53 (2H, t), 4.60 (2H, s), 7.06 (2H, d), 7.15 (2H, d), 7.46(1H, d), 7.59 (1H, q), 7.98 (1H, d), 8.02 (1H, d), 8.51 (1H, d).

Example B184 A Mixture of2-Benzoyl-5-bromo-1,2-dihydro-1-isoquinolinecarbonitrile and2-Benzoyl-7-bromo-1,2-dihydro-1-isoquinolinecarbonitrile

[0863]

[0864] The title compounds were obtained by treating 5- or7-bromoisoquinoline, which was synthesized according to J. Am. Chem.Soc., 61, 183 (1939), in the same manner as in Example B140. Theobtained compounds were used in the following reaction withoutseparation and purification.

Example B185 7-Bromo-1-(4-butylbenzyl)isoquinoline

[0865]

[0866] The title compound was obtained by treating the compound ofExample B184 and the compound of Example B1 in the same manner as inExample B2.

[0867]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.28-1.37 (2H, m), 1.51-1.58(2H, m), 2.55 (2H, t), 4.58 (2H, s), 7.09 (2H, d), 7.18 (2H, d),7.51-7.53 (1H, m), 7.69-7.70 (2H, m), 8.33-8.34 (1H, m), 8.52 (1H, d).

Example B186 5-Benzoyl-4,5-dihydrothieno[3,2-c]pyridine-4-carbonitrile

[0868]

[0869] The title compound was obtained by treatingthieno[3,2-c]pyridine, synthesized according to J. Heterocycl. Chem.,30, 183 (1993), in the same manner as in Example B140.

[0870]¹H-NMR (CDCl₃) δ (ppm): 6.05 (1H, d), 6.57 (1H, brd), 6.66 (1H,s), 7.07 (1H, d), 7.32 (1H, d), 7.46-7.50 (2H, m), 7.54-7.62 (3H, m).

Example B187 4-(4-Butylbenzyl)thieno[3,2-c]pyridine

[0871]

[0872] The title compound was obtained by treating the compound ofExample B186 and the compound of Example B1 in the same manner as inExample B2.

[0873]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.27-1.37 (2H, m), 1.51-1.59(2H, 1.5 m), 2.54 (2H, t), 4.47 (2H, s), 7.07 (2H, d), 7.19 (2H, d),7.42 (1H, d), 7.47 (1H, dd), 7.68 (1H, d), 8.41 (1H, d).

Example B188 4-(4-Methoxybenzyl)thieno[3,2-c]pyridine

[0874]

[0875] The title compound was obtained by treating the compound ofExample B186 and 4-methoxybenzyl chloride in the same manner as inExample B2.

[0876]¹H-NMR (CDCl₃) δ (ppm): 3.75 (3H, s), 4.44 (2H, s), 6.79-6.82 (2H,m), 25 7.19-7.22 (2H, m), 7.43 (1H, d), 7.46 (1H, dd), 7.68 (1H, d),8.41 (1H, d).

Example B189 4-(Thieno[3,2-c]pyridin-4-ylmethyl)phenylTrifluoromethanesulfonate

[0877]

[0878] A solution of boron tribromide in methylene chloride (1.0 M, 10ml, 10 mmol) was added dropwise to a solution of the compound of ExampleB188 (510 mg, 2.0 mmol) in methylene chloride (10 ml)cooled to 0° C. ,and this reaction mixture was stirred at that temperature for 1.5 hours.The reaction mixture was made weakly alkaline by addition of a saturatedaqueous sodium hydrogencarbonate solution, extracted with ethyl acetate,dried over anhydrous magnesium sulfate, and then concentrated underreduced pressure. The obtained residue was dissolved in pyridine, andthe resulting solution was cooled to 0° C. Aftertrifluoromethanesulfonic anhydride (0.34 ml, 2.1 mmol) was addeddropwise thereto, the mixture was stirred at that temperature for 2hours, poured on ice, extracted with ethyl acetate, dried over anhydrousmagnesium sulfate, and then concentrated under reduced pressure. Theresidue was purified by silica gel chromatography to give the titlecompound (312 mg).

[0879]¹H-NMR (CDCl₃) δ (ppm): 4.52 (2H, s), 7.16-7.18 (2H, m), 7.36 (2H,m), 7.43-7.44 (1H, m), 7.49 (1H, d), 7.73 (1H, d), 8.42 (1H, d).

Example B190 4-(4-Bromobenzyl)thieno[3,2-c]pyridine

[0880]

[0881] The title compound was obtained by treating the compound ofExample B186 and the compound of Example B31 in the same manner as inExample B2.

[0882]¹H-NMR (CDCl₃) δ (ppm): 4.45 (2H, s), 7.14-7.16 (2H, m), 7.37-7.39(2H, m), 7.41-7.43 (1H, m), 7.45 (1H, d), 7.71 (1H, d), 8.41 (1H, d).

Example B191 4-(4-Bromo-2-fluorobenzyl)thieno[3,2-c]pyridine

[0883]

[0884] The title compound was obtained by treating the compound ofExample B186 and 4-bromo-2-fluorobenzyl bromide in the same manner as inExample B2.

[0885]¹H-NMR (CDCl₃) δ (ppm): 4.46 (2H, s), 7.11 (1H, t), 7.15-7.18 (1H,m), 7.22-7.25 (1H, m), 7.47 (1H, d), 7.49 (1H, d), 7.71 (1H, d), 8.41(1H, d).

Example B1924-{4-[4-(Tetrahydro-2H-2-pyranyloxy)-1-butynyl]benzyl}thieno[3,2-c]pyridine

[0886]

[0887] The title compound was obtained by treating the compound ofExample B189 and 2-(3-butynyloxy)tetrahydro-2H-pyran in the same manneras in Example B42.

[0888]¹H-NMR (CDCl₃) δ (ppm): 1.40-1.90 (6H, m), 2.69 (2H, t), 3.45-3.65(2H, m), 3.78-3.95 (2H, m), 4.48 (2H, s), 4.66-4.69 (1H, m), 7.18 (2H,d), 7.27 (2H, d), 7.41 (1H, d), 7.44 (1H, d), 7.70 (1H, d), 8.41 (1H,d).

Example B193 4-[4-(Thieno[3,2-c]pyridin-4-ylmethyl)phenyl]-3-butyn-1-ol

[0889]

[0890] The title compound was obtained by treating the compound ofExample B192 in the same manner as in Example B47.

[0891]¹H-NMR (CDCl₃) δ (ppm): 2.67 (2H, t), 3.79 (2H, t), 4.50 (2H, s),7.20 (2H, d), 7.32 (2H, d), 7.41 (1H, d), 7.44 (1H, d), 7.71 (1H, d),8.42 (1H, d).

[0892] The proton of the hydroxyl group was not observed in the NMRspectrum.

Example B194 6-Benzoyl-6,7-dihydrothieno[2,3-c]pyridine-7-carbonitrile

[0893]

[0894] The title compound was obtained by treatingthieno[2,3-c]pyridine, which was synthesized according to J. Heterocycl.Chem., 30, 183 (1993), in the same manner as in Example B140.

[0895]¹H-NMR (CDCl₃) δ (ppm): 6.07 (1H, d), 6.56 (1H, brd), 6.75 (1H,s), 6.97 (1H, d), 7.37 (1H, d), 7.46-7.51 (2H, m), 7.54-7.64 (3H, m).

Example B195 7-(4-Butylbenzyl)thieno[2,3-c]pyridine

[0896]

[0897] The title compound was obtained by treating the compound ofExample B194 and the compound of Example B1 in the same manner as inExample B2.

[0898]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.28-1.37 (2H, m), 1.51-1.59(2H, m), 2.55 (2H, t), 4.40 (2H, s), 7.09 (2H, d), 7.28 (2H, d), 7.34(1H, d), 7.57 (1H, d), 7.62 (1H, d), 8.47 (1H, d).

Example B196 7-(4-Methoxybenzyl)thieno[2,3-c]pyridine

[0899]

[0900] The title compound was obtained by treating the compound ofExample B194 and 4-methoxybenzyl chloride in the same manner as inExample B2.

[0901]¹H-NMR (CDCl₃) δ (ppm): 3.76 (3H, s), 4.38 (2H, s), 6.81-6.83 (2H,m), 7.28-7.30 (2H, m), 7.35 (1H, d), 7.57 (1H, d), 7.62 (1H, d), 8.47(1H, d).

Example B197 4-(Thieno[2,3-c]pyridin-7-ylmethyl)phenylTrifluoromethanesulfonate

[0902]

[0903] The title compound was obtained by treating the compound ofExample B196 in the same manner as in Example B189.

[0904]¹H-NMR (CDCl₃) δ (ppm): 4.44 (2H, s), 7.17-7.19 (2H, m), 7.38-7.40(1H, m), 7.44-7.46 (2H, m), 7.61 (1H, d), 7.65-7.67 (1H, m), 8.47-8.49(1H, m).

Example B198 7-(4-Bromobenzyl)thieno[2,3-c]pyridine

[0905]

[0906] The title compound was obtained by treating the compound ofExample B194 and the compound of Example B31 in the same manner as inExample B2.

[0907]¹H-NMR (CDCl₃) δ (ppm): 4.37 (2H, s), 7.23-7.25 (2H, m), 7.37 (1H,d), 7.39-7.41 (2H, m), 7.59 (1H, d), 7.63-7.65 (1H, m), 8.47 (1H, d).

Example B199 7-(4-Bromo-2-fluorobenzyl)thieno[2,3-c]pyridine

[0908]

[0909] The title compound was obtained by treating the compound ofExample B194 and 4-bromo-2-fluorobenzyl bromide in the same manner as inExample B2.

[0910]¹H-NMR (CDCl₃) δ (ppm): 4.40-4.41 (2H, m), 7.12-7.20 (2H, m),7.23-7.26 (1H, m), 7.37-7.39 (1H, m), 7.59-7.62 (1H, m), 7.65-7.67 (1H,m), 8.45-8.47 (1H, m).

Example B2007-{4-[4-(Tetrahydro-2H-2-pyranyloxy)-1-butynyl]benzyl}thieno[2,3-c]pyridine

[0911]

[0912] The title compound was obtained by treating the compound ofExample B197 and 2-(3-butynyloxy)tetrahydro-2H-pyran in the same manneras in Example B42.

[0913]¹H-NMR (CDCl₃) δ (ppm): 1.50-1.90 (6H, m), 2.69 (2H, t), 3.49-3.54(1H, m), 3.58-3.65 (1H, m), 3.85-3.95 (2H, m), 4.41 (2H, s), 4.68 (1H,t), 7.26-7.31 (4H, m), 7.36 (1H, d), 7.58 (1H, d), 7.63 (1H, d), 8.47(1H, d).

Example B201 4-[4-(Thieno[2,3-c]pyridin-7-ylmethyl)phenyl]-3-butyn-1-ol

[0914]

[0915] The title compound was obtained by treating the compound ofExample B200 in the same manner as in Example B47.

[0916]¹H-NMR (CDCl₃) δ (ppm): 1.99 (1H, brs), 2.67 (2H, t), 3.79 (2H,t), 4.42 (2H, s), 7.27-7.34 (4H, m), 7.36 (1H, d), 7.59 (1H, d), 7.64(1H, d), 8.47 (1H, d).

Example B202 2-Chloro-3-(methoxymethoxy)pyridine

[0917]

[0918] Sodium hydride (66%, 633 mg, 17.4 mmol) was added to anice-cooled solution of 2-chloro-3-hydroxypyridine (2.05 g, 15.8 mmol) intetrahydrofuran (30 ml) under nitrogen atmosphere, and this reactionmixture was stirred at that temperature for 15 minutes. Chloromethylmethyl ether (1.32 ml, 17.4 mmol) was added, and the resulting reactionmixture was stirred at that temperature for 30 minutes, then at roomtemperature for another 2 hours. After water was added, the reactionmixture was extracted with ethyl acetate, washed with saturated brine,and then concentrated under reduced pressure. The residue was purifiedby silica gel column chromatography to give the title compound (2.44 g).

[0919]¹H-NMR (CDCl₃) δ (ppm): 3.53 (3H, s), 5.28 (2H, s), 7.19 (1H, dd),7.49 (1H, dd), 8.06 (1H, dd).

Example B203 2-Chloro-4-iodo-3-(methoxymethoxy)pyridine

[0920]

[0921] A solution of the compound of Example B202 (1.40 g, 8.06 mmol) indiethyl ether (8 ml) was added dropwise to a solution of 1.51 Mt-butyllithium-n-pentane solution (8.01 ml, 12.1 mmol) in diethyl ether(15 ml) cooled to −78° C. under nitrogen atmosphere, and the reactionmixture was stirred at that temperature for 15 minutes. After iodine(3.07 g, 12.1 mmol) was added, the reaction mixture was gradually warmedto room temperature. An aqueous sodium thiosulfate solution was furtheradded, and the diethyl ether layer was separated, washed with saturatedbrine, and then concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compound(356 mg).

[0922]¹H-NMR (CDCl₃) δ (ppm): 3.73 (3H, s), 5.22 (2H, s), 7.69 (1H, d),7.80 (1H, d).

Example B204 7-Chlorofuro[2,3-c]pyridine

[0923]

[0924] Trimethylsilylacetylene (28.3 μl, 0.201 mmol) and triethylamine(59.8 μl, 0.429 mmol) were added to a solution of the compound ofExample B203 (36.6 mg, 0.143 mmol),tetrakis(triphenylphosphine)palladium (16.5 mg, 0.0143 mmol), andcopper(I) iodide (2.7 mg, 0.014 mmol) in dimethylformamide (1.5 ml), andthis mixture was stirred at 50° C. for 4 hours. After allowing themixture to cool to room temperature, water was added thereto, and theresulting mixture was extracted with ethyl acetate, washed withsaturated brine, and then concentrated under reduced pressure. Theresidue was dissolved in methanol (5 ml), potassium carbonate (100 mg,0.724 mmol) was added thereto, and the resulting mixture was stirred atroom temperature for 1 hour. After water was added, the mixture wasextracted with diethyl ether, washed with saturated brine, and thenconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound (5.5 mg).

[0925]¹H-NMR (CDCl₃) δ (ppm): 6.89 (1H, d), 7.51 (1H, d), 7.83 (1H, d),8.21 (1H, d).

Example B205 4-Butylbenzylmagnesium Chloride

[0926]

[0927] A mixed solution of the compound of Example B1 (1.04 g, 5.69mmol), magnesium (761 mg, 31.3 mmol), and a catalytic amount of1,2-dibromoethane in diethyl ether (11 ml) was initiated by heatingunder reflux. After the heat source was removed, a solution of thecompound of Example B1 (4.16 g, 22.8 mmol) in diethyl ether (60 ml) wasadded dropwise to the reaction mixture at a rate that maintains gentlereflux, and the mixture was heated under reflux for 30 minutes. Themixture was then allowed to cool to room temperature to give the titlecompound as a 0.4 M solution in diethyl ether. This solution was used inthe following reaction as it is.

Example B206 7-(4-Butylbenzyl)furo[2,3-c]pyridine

[0928]

[0929] The compound of Example B205 (300 μl, 0.1 mmol) was added to asolution of the compound of Example B204 (5.0 mg, 0.033 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloronickel(II) (4.5 mg, 0.0065mmol) in tetrahydrofuran (1 ml), and the mixture was stirred at 50° C.for 1 hour. After allowing the mixture to cool to room temperature,ethyl acetate was added thereto. The resulting mixture was washed with asaturated aqueous ammonium chloride solution and saturated brine, thenconcentrated under reduced pressure. The residue was purified byNH-silica gel column chromatography to give the title compound (2.9 mg).

[0930]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.29-1.35 (2H, m), 1.50-1.58(2H, m), 2.54 (2H, t), 4.40 (2H, s), 6.78 (1H, d), 7.08 (2H, d), 7.30(2H, d), 7.40 (1H, d), 7.72 (1H, d), 8.34 (1H, d).

Example B207 7-(4-Butylbenzyl)-1H-pyrrolo[2,3-c]pyridine

[0931]

[0932] The compound of Example B205 (800 μl, 0.3 mmol) was added to asolution of 1-chloropyrrolopyridine (19.4 mg, 0.127 mmol), which wassynthesized from 2-chloro-3-aminopyridine according to the method ofH07-165,708A, and dichloro(diphenylphosphinopropane) nickel (6.9 mg,0.013 mmol) in tetrahydrofuran (1 ml) under ice-cooling, and the mixturewas stirred while heating under reflux for 4 hours. After allowing themixture to cool to room temperature, ethyl acetate was added thereto.The resulting mixture was washed with a saturated aqueous ammoniumchloride solution and saturated brine, then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (7.1 mg).

[0933]¹H-NMR (CDCl₃) δ (ppm): 0.91 (3H, t), 1.31-1.37 (2H, m), 1.55-1.59(2H, m), 2.58 (2H, t), 4.44 (2H, s), 6.50 (1H, d), 7.12 (2H, d), 7.18(1H, d), 7.22 (2H, d), 7.45 (1H, d), 8.21 (1H, d).

[0934] The NH proton was not observed in the NMR spectrum.

Example B208 4-(4-Butylbenzyl)-1-imidazo[4,5-c]pyridine

[0935]

[0936] The compound of Example B205 (3.45 ml, 1.38 mmol) was added to asolution of 1-chloroimidazopyridine (88.6 mg, 0.577 mmol), which wassynthesized from 4-amino-2-chloropyridine according to the methoddescribed in J. Heterocycl. Chem., 2, 196 (1965), anddichloro(diphenylphosphinopropane)nickel (31.3 mg, 0.0577 mmol) intetrahydrofuran (2 ml), and the mixture was stirred while heating underreflux for 2 hours. After allowing the mixture to cool to roomtemperature, ethyl acetate was added thereto. The resulting mixture wasfiltered through silica gel and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to give thetitle compound (64.2 mg).

[0937]¹H-NMR (CDCl₃) δ (ppm): 0.86 (3H, t), 1.23-1.32 (2H, m), 1.44-1.52(2H, m), 2.47 (2H, t), 4.56 (2H, s), 7.02 (2H, d), 7.19 (2H, d), 7.34(1H, d), 8.00 (1H, s), 8.25-8.27 (1H, m).

[0938] The NH proton was not observed in the NMR spectrum.

Example B209 4-Bromo-1-isoquinolinol

[0939]

[0940] Bromine (1.78 ml, 34.5 mmol) was added to an ice-cooled solutionof 1-hydroxyisoquinoline (5.01 g, 34.5 mmol) in acetic acid (50 ml) andthis reaction mixture was stirred at room temperature for 2 hours.Water, ethyl acetate, and tetrahydrofuran were added, and the resultingreaction mixture was filtered through filter paper. The organic layerwas washed with saturated brine and concentrated under reduced pressure.The residue was recrystallized from ethyl acetate and hexane to give thetitle compound (6.19 g).

[0941]¹H-NMR (DMSO-d6) δ (ppm): 7.56 (1H, s), 7.59-7.63 (1H, m),7.76-7.78 (1H, m), 7.84-7.89 (1H, m), 8.23-8.26 (1H, m), 11.59 (1H, brs).

Example B210 1,4-Dibromoisoquinoline

[0942]

[0943] A mixed solution of the compound of Example B209 (1.40 g, 8.06mmol) and phosphorus tribromide (6 ml) was stirred at 150° C. for 1hour, and then heated under reflux for another 1 hour. The reactionmixture was allowed to cool to room temperature, poured on ice, thenwarmed to room temperature. Ethyl acetate was added, and the resultingmixture was washed with saturated brine and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (845 mg).

[0944]¹H-NMR (CDCl₃) δ (ppm): 7.76-7.80 (1H, m), 7.86-7.90 (1H, m), 8.19(1H, d), 8.31-8.34 (1H, m), 8.48 (1H, s).

Example B211 4-Bromo-1-(4-butylbenzyl)isoquinoline

[0945]

[0946] The compound of Example B205 (2.5 ml, 1 mmol) was added to asolution of the compound of Example B210 (200 mg, 0.697 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloronickel(II) (75.6 mg, 0.139mmol) in tetrahydrofuran (2 ml), and the mixture was stirred at roomtemperature for 30 minutes. After ethyl acetate was added, the resultingmixture was washed successively with a saturated aqueous ammoniumchloride solution, a saturated aqueous sodium hydrogencarbonatesolution, and saturated brine, then concentrated under reduced pressure.The residue was purified by silica gel column chromatography to give thetitle compound (98 mg).

[0947]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.29-1.34 (2H, m), 1.51-1.60(2H, m), 2.53 (2H, t), 4.59 (2H, s), 7.06 (2H, d), 7.16 (2H, d),7.57-7.61 (1H, m), 7.73-7.77 (1H, m), 8.15-8.19 (2H, m), 8.69 (1H, s).

Example B212 1-(4-Butylbenzyl)-5,6,7,8-tetrahydroisoquinoline

[0948]

[0949] The compound of Example B211 (13.0 mg, 0.0367 mmol) was dissolvedin a mixed solution of ethyl acetate and methanol (1:1, 1 ml), 10%palladium-carbon (containing 50% water, 13 mg) was added, and themixture was stirred at room temperature under hydrogen atmosphere atatmospheric pressure for 12 hours. After purging the reaction systemwith nitrogen, the catalyst was removed by filtration through celite.The obtained filtrate was concentrated under reduced pressure to givethe title compound (8.8 mg).

[0950]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.28-1.38 (2H, m), 1.52-1.59(2H, m), 1.74-1.82 (4H, m), 2.55 (2H, t), 2.66 (2H, t), 2.81 (2H, t),4.26 (2H, s), 7.07-7.15 (5H, m), 8.32 (1H, d).

Example B213 1-[2-(Phenyl)benzyl]isoquinoline

[0951]

[0952] The title compound was obtained by treating 2-phenylbenzylbromide instead of n-butylbenzyl chloride in the same manner as inExample B2.

[0953]¹H-NMR (CDCl₃) δ (ppm): 4.62 (2H, s), 7.05 (1H, d), 7.16 (1H, dd),7.22-7.50 (8H, m), 7.52 (1H, d), 7.58 (1H, dd), 7.65 (1H, d), 7.76 (1H,d), 8.47 (1H, d).

Example B214 1-[4-Fluoro-2-(trifluoromethyl)benzyl]isoquinoline

[0954]

[0955] The title compound was obtained by treating4-fluoro-2-(trifluoromethyl)benzyl methanesulfonate instead ofn-butylbenzyl chloride in the same manner as in Example B2.

[0956]¹H-NMR (CDCl₃) δ (ppm): 4.83 (2H, s), 6.87 (1H, dd), 7.01 (1H,ddd), 7.43 (1H, dd), 7.54 (1H, dd), 7.61 (1H, d), 7.67 (1H, dd), 7.85(1H, d), 7.96 (1H, d), 8.49 (1H, d).

Example B215 1,3-Benzodioxoyl-4-yl-(1-isoquinolyl)methanol

[0957]

[0958] The title compound was obtained bytreating2,3-methylenedioxybenzaldehyde in the same manner as in ExampleB82.

[0959]¹H-NMR (CDCl₃) δ (ppm): 5.97-5.99 (1H, m), 6.09 (1H, brs),6.20-6.40 (1H, m), 6.54-6.60 (2H, m), 6.65-6.70 (2H, m), 7.52 (1H, dd),7.63 (1H, d), 7.64 (1H, dd), 7.84 (1H, d), 8.04 (1H, d), 8.53 (1H, d).

Example B216 1,3-Benzodioxoyl-4-yl-(1-isoquinolyl)methyl Acetate

[0960]

[0961] The title compound was obtained by treating the compound ofExample B215 in the same manner as in Example B38.

[0962]¹H-NMR (CDCl₃) δ (ppm): 2.23 (3H, s), 5.98-6.02 (2H, m), 6.74-6.79(1H, m), 6.90-6.93 (1H, m), 7.15-7.19 (1H, m), 7.23-7.28 (1H, m), 7.58(1H, dd), 7.60 (1H, d), 7.66 (1H, dd), 7.83 (1H, d), 8.28 (1H, d), 8.57(1H, d).

Example B217 1-(1,3-Benzodioxoyl-4-ylmethyl)isoquinoline

[0963]

[0964] The title compound was obtained by treating the compound ofExample B216 in the same manner as in Example B39.

[0965]¹H-NMR (CDCl₃) δ (ppm): 4.62 (2H, s), 6.02 (2H, s), 6.64-6.70 (3H,m), 7.57 (1H, dd), 7.58 (1H, d), 7.66 (1H, dd), 7.83 (1H, d), 8.23 (1H,d), 8.50 (1H, d).

Example B218 1-(1-Naphthylmethyl)isoquinoline

[0966]

[0967] The title compound was obtained by treating1-(chloromethyl)naphthalene instead of n-butylbenzyl chloride in thesame manner as in Example B2.

[0968]¹H-NMR (CDCl₃) δ (ppm): 5.13 (2H, s), 6.96 (1H, d), 7.29 (1H, d),7.45-7.67 (5H, m), 7.72 (1H, d), 7.84-7.90 (2H, m), 8.08 (1H, d), 8.26(1H, d), 8.52 (1H, d).

Example B219 3-Bromophenylbutyrate

[0969]

[0970] n-Butyryl chloride (7.25 ml) was added to an ice-cooled solutionof 3-bromophenol (10.0 g) in pyridine (50 ml), and this reaction mixturewas stirred at that temperature for 3 hours, then at room temperaturefor another 3.5 hours. After ice was added, the reaction mixture wasextracted with ethyl acetate, washed with 1 N hydrochloric acid andwater, dried over anhydrous magnesium sulfate, and then concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (12.77 g).

[0971]¹H-NMR (CDCl3) δ (ppm): 1.04 (3H, t), 1.72-1.82 (2H, m), 2.54 (2H,t), 7.04 (1H, dd), 7.22-7.29 (2H, m), 7.36 (1H, d).

Example B220 1-(4-Bromo-2-hydroxyphenyl)-1-butanone

[0972]

[0973] Aluminum chloride (10.51 g) was added to a solution of thecompound of Example B219 (12.77 g) in chlorobenzene (70 ml) undernitrogen atmosphere, and this reaction mixture was stirred while heatingunder reflux for 9 hours. After the reaction mixture was cooled to roomtemperature, ice was added thereto. The resulting mixture was extractedwith ethyl acetate, washed with water, dried over anhydrous magnesiumsulfate, and then concentrated under reduced pressure. The compound thusobtained was used in the following reaction without furtherpurification.

[0974]¹H-NMR (CDCl3) δ (ppm): 0.91 (3H, t), 1.53-1.65 (2H, m), 3.00 (2H,t), 7.02 (1H, dd), 7.19 (1H, d), 7.78 (1H, d), 12.50 (1H, s).

Example B221 1-(4-Bromo-2-methoxyphenyl)-1-butanone

[0975]

[0976] Potassium carbonate (9.07 g) and methyl iodide (3.92 ml) wereadded to a solution of the compound of Example B220 (13.30 g) in acetone(75 ml), and this reaction mixture was stirred while heating underreflux for 4 hours. The reaction mixture was filtered through celite,ether was added to remove insoluble material by filtration, and thefiltrate was concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compound(9.52 g).

[0977]¹H-NMR (CDCl3) δ (ppm): 0.95 (3H, t), 1.64-1.74 (2H, m), 2.91 (2H,t), 3.90 (3H, s), 7.10 (1H, d), 7.14 (1H, dd), 7.54 (1H, d).

Example B222 4-Bromo-1-butyl-2-methoxybenzene

[0978]

[0979] The title compound was obtained by treating the compound ofExample B221 in the same manner as in Example B3.

[0980]¹H-NMR (CDCl3) δ (ppm): 0.92 (3H, t), 1.29-1.39 (2H, m), 1.48-1.56(2H, m), 2.54 (2H, t), 3.81 (3H, s), 6.95 (1H, s), 6.96-7.02 (2H, m).

Example B223 (4-Butyl-3-methoxyphenyl)(1-isoquinolyl)ketone

[0981]

[0982] A mixture containing the title compound was obtained by treatingthe compound of Example B222 in the same manner as in Example B36.

[0983] This mixture was used in the following reaction withoutseparation and purification.

Example B224 (4-Butyl-3-methoxyphenyl)(1-isoquinolyl)methanol

[0984]

[0985] A mixture containing the title compound was obtained by treatingthe compound of Example B223 in the same manner as in Example B37.

[0986] This mixture was used in the following reaction withoutseparation and purification.

Example B225 (4-Butyl-3-methoxyphenyl)(1-isoquinolyl)methyl Acetate

[0987]

[0988] The title compound was obtained by treating the compound ofExample B224 in the same manner as in Example B38.

[0989]¹H-NMR (CDCl3) δ (ppm): 0.90 (3H, t), 1.24-1.38 (2H, m), 1.46-1.60(2H, m), 2.24 (3H, s), 2.54 (2H, t), 3.76 (3H, s), 6.97 (1H, s), 6.98(1H, d), 7.06 (1H, d), 7.53-7.67 (4H, m), 7.83 (1H, d), 8.26 (1H, d),8.58 (1H, d).

Example B226 1-(4-Butyl-3-methoxybenzyl)isoquinoline

[0990]

[0991] The title compound was obtained by treating the compound ofExample B225 in the same manner as in Example B39.

[0992]¹H-NMR (CDCl3) δ (ppm): 0.89 (3H, t), 1.27-1.38 (2H, t), 1.45-1.54(2H, t), 2.52 (2H, t), 3.72 (3H, s), 4.63 (2H, s), 6.78 (1H, d), 6.79(1H, s), 6.99 (1H, d), 7.53 (1H, dd), 7.55 (1H, d), 7.64 (1H, dd), 7.80(1H, d), 8.19 (1H, d), 8.49 (1H, d).

Example B227 2-Butyl-5-(1-isoquinolylomethyl)phenol

[0993]

[0994] The title compound was obtained by treating the compound ofExample B226 in the same manner as in Example B40.

[0995]¹H-NMR (CDCl3) δ (ppm): 0.91 (3H, t), 1.30-1.40 (2H, m), 1.52-1.65(2H, m), 2.55 (2H, t), 4.55 (2H, s), 6.46 (1H, brs), 6.85 (1H, d), 7.03(1H, d), 7.32-7.40 (1H, m), 7.55 (1H, dd), 7.68 (1H, dd), 7.81 (1H, d),7.94-8.05 (1H, m), 8.14 (1H, d).

[0996] The proton of the phenolic hydroxyl group was not observed in theNMR spectrum.

Example B228 2-Bromo-3-(methoxymethoxy)pyridine

[0997]

[0998] The title compound was synthesized in the same manner as inExample B202 by using 2-bromo-3-hydroxypyridine.

[0999]¹H-NMR (CDCl₃) δ (ppm): 3.53 (3H, s), 5.29 (2H, s), 7.19-7.23 (1H,m), 7.42-7.45 (1H, m), 8.04-8.06 (1H, m).

Example B229 2-(4-Butylbenzyl)-3-(methoxymethoxy)pyridine

[1000]

[1001] The compound of Example B205 (7 ml, 3 mmol) was added to anice-cooled mixed solution of the compound of Example B228 (524 mg, 2.40mmol) and dichloro(diphenylphosphinopropane)nickel (65.0 mg, 0.120 mmol)in tetrahydrofuran (10 ml), and the mixture was stirred while heatingunder reflux for 5 hours. After allowing the mixture to cool to roomtemperature, ethyl acetate was added. The resulting mixture was washedsuccessively with a saturated aqueous ammonium chloride solution, asaturated aqueous sodium hydrogencarbonate solution, and saturatedbrine, then concentrated under reduced pressure. The residue wasfiltered through NH-silica gel. After concentrating under reducedpressure, the residue was dissolved in methanol (15 ml), triethylamine(500 μl, 3.59 mmol) and 10% palladium-carbon (containing 50% water, 50mg) were added, and the resulting mixture was stirred at roomtemperature under hydrogen atmosphere at atmospheric pressure for 3hours. After purging the reaction system with nitrogen, the catalyst wasremoved by filtration through celite, and the filtrate was concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (280 mg).

[1002]¹H-NMR (CDCl₃) δ (ppm): 0.89 (3H, t), 1.28-1.34 (2H, m), 1.52-1.58(2H, m), 2.53 (2H, t), 3.33 (3H, s), 4.16 (2H, s), 5.16 (2H, s),7.04-7.10 (3H, m), 7.20 (2H, d), 7.33-7.35 (1H, m), 8.19-8.20 (1H, m).

Example B230 2-(4-Butylbenzyl)-3-pyridinol

[1003]

[1004] Trifluoroacetic acid (1 ml) was added to a solution of thecompound of Example B229 (256 mg, 0.849 mmol) in methylene chloride (5ml), and this reaction mixture was stirred at room temperatureovernight. After a saturated aqueous sodium hydrogencarbonate solutionand ethyl acetate were added, the reaction mixture was washed withsaturated brine and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compound(182 mg).

[1005]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.28-1.37 (2H, m), 1.51-1.58(2H, m), 2.54 (2H, t), 4.20 (2H, s), 7.02-7.08 (4H, m), 7.22 (2H, d),8.08-8.09 (1H, m).

[1006] The proton of the phenolic hydroxyl group was not observed in theNMR spectrum.

Example B231 2-(4-Butylbenzyl)-3-methoxypyridine

[1007]

[1008] Potassium carbonate (33.0 mg, 0.239 mmol) and methyl iodide (14.9μl, 0.239 mmol) were added to a solution of the compound of Example B230(19.2 mg, 0.0796 mmol) in acetone (1 ml), and this reaction mixture wasstirred at room temperature for 3 hours. After ethyl acetate was added,the reaction mixture was washed with saturated brine and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (1.47 mg).

[1009]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.32-1.34 (2H, m), 1.53-1.57(2H, m), 2.54 (2H, t), 3.82 (3H, s), 4.14 (2H, s), 7.06 (2H, d),7.10-7.11 (2H, m), 7.21 (2H, d), 8.12-8.14 (1H, m).

Example B232 2-(4-Butylbenzyl)-3-chloropyridine

[1010]

[1011] The compound of Example B205 (12 ml, 5 mmol) was added to anice-cooled mixed solution of 2,3-dichloropyridine (525 mg, 3.55 mmol)and dichloro(diphenylphosphinopropane)nickel (96.2 mg, 0.178 mmol) intetrahydrofuran (4 ml), and this reaction mixture was stirred at roomtemperature for 1 hour. After ethyl acetate was added, the reactionmixture was washed successively with a saturated aqueous ammoniumchloride solution, a saturated aqueous sodium hydrogencarbonatesolution, and saturated brine, and then concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (199 mg).

[1012]¹H-NMR (CDCl₃) δ (ppm): 0.91 (3H, t), 1.29-1.38 (2H, m), 1.52-1.60(2H, m), 2.56 (2H, t), 4.28 (2H, s), 7.08-7.13 (3H, m), 7.21 (2H, d),7.64 (1H, dd), 8.46 (1H, dd).

Example B233 2-(4-Butylbenzyl)-3-ethylpyridine

[1013]

[1014] Ethylmagnesium chloride (0.97 M, 102 μl, 0.993 mmol) was added toa mixed solution of the compound of Example B232 (12.9 mg, 0.0496 mmol)and dichloro(diphenylphosphinoferrocene)nickel (3.4 mg, 0.0050 mmol) intetrahydrofuran (1 ml). The reaction mixture was stirred at 50° C. for 1hour, then heated under reflux for another 2 hours. After allowing thereaction mixture to reach room temperature, ethyl acetate was addedthereto. The reaction mixture was washed with a saturated aqueousammonium chloride solution and saturated brine, then concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound (3.29 mg).

[1015]¹H-NMR (CDCl₃) δ (ppm): 0.90-0.93 (6H, m), 1.30-1.37 (2H, m),1.54-1.59 (2H, m), 2.55-2.59 (4H, m), 4.12 (2H, s), 7.05-7.18 (5H, m),7.55-7.59 (1H, m), 8.53-8.55 (1H, m).

Example B234

[1016] tert-Butyl N-(2-Bromo-3-pyridyl)carbamate

[1017] N-bromosuccinimide (7.51 g, 42.2 mmol) was added to an ice-cooledmixed solution of 3-aminopyridine (3.97 g, 42.2 mmol) indimethylformamide (25 ml), and this reaction mixture was stirred at thattemperature for 30 minutes. After ethyl acetate was added, the reactionmixture was washed with saturated brine and concentrated under reducedpressure. A solution of the residue in methylene chloride (20 ml) wascooled on ice, then triethylamine (3.74 ml, 26.8 mmol), a catalyticamount of dimethylaminopyridine, and di-t-butyl dicarbonate (3.08 ml,13.4 mmol) were added to the solution, and the mixture was stirred atroom temperature overnight. After concentration under reduced pressure,the residue was purified by silica gel column chromatography to give thetitle compound (344 mg).

[1018]¹H-NMR (CDCl₃) δ (ppm): 1.55 (9H, s), 7.03 (1H, brs), 7.25 (1H,dd), 8.03 (1H, dd), 8.46 (1H, d).

Example B235 2-Bromo-3-(N-t-butoxycarbonyl-N-methyl)aminopyridine

[1019]

[1020] Methyl iodide (157 μl, 2.52 mmol) and 66% sodium hydride (91.6mg, 2.52 mmol) were added to an ice-cooled solution of the compound ofExample B234 (344 mg, 1.26 mmol) in dimethylformamide (5 ml), and thisreaction mixture was stirred at that temperature for 40 minutes. Afterethyl acetate was added, the reaction mixture was washed with saturatedbrine and filtered through silica gel. The organic layer wasconcentrated under reduced pressure to give the title compound (356 mg).

[1021]¹H-NMR (CDCl₃) δ (ppm): 1.36 (9H, s), 3.17 (3H, s), 7.30 (1H, dd),7.55 (1H, d), 8.30 (1H, dd).

Example B236 N-[2-(4-Butylbenzyl)-3-pyridyl]-N-methylamine

[1022]

[1023] To a methylene chloride solution (2 ml) of a compound, which wasobtained by introduction of a 4-butylbenzyl group to the compound ofExample B235 (62.8 mg, 0.219 mmol) in the same manner as in ExampleB211, trifluoroacetic acid (2 ml) was added. The mixture was stirred atroom temperature for 1 hour, and then added dropwise to an aqueoussolution of sodium hydrogencarbonate. After ethyl acetate was added, themixture was washed with saturated brine and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound (29.7 mg).

[1024]¹H-NMR (CDCl₃) δ (ppm): 0.91 (3H, t), 1.29-1.38 (2H, m), 1.53-1.60(2H, m), 2.56 (2H, t), 2.72 (3H, s), 3.63 (1H, br s), 4.09 (2H, s), 6.86(1H, d), 7.08-7.12 (5H, m), 7.98 (1H, dd).

Example B237 N-[2-(4-Butylbenzyl)-3-pyridyl]-N,N-dimethylamine

[1025]

[1026] Acetic acid (12.1 μl, 0.211 mmol), 37% formalin (15.8 μl, 0.211mmol), and sodium triacetoxyborohydride (44.7 mg, 0.211 mmol) were addedto an ice-cooled solution of the compound of Example B236 (26.8 mg,0.105 mmol) in methylene chloride (2 ml), and the mixture was stirred atroom temperature for 30 minutes. After ethyl acetate was added, themixture was washed with a saturated aqueous sodium hydrogencarbonatesolution and saturated brine and concentrated under reduced pressure.The residue was purified by silica gel column chromatography to give thetitle compound (23.3 mg)

[1027]¹H-NMR (CDCl₃) δ (ppm): 0.91 (3H, t), 1.30-1.36 (2H, m), 1.52-1.59(2H, m), 2.55 (2H, t), 2.67 (6H, s), 4.24 (2H, s), 7.06 (2H, d), 7.10(1H, dd), 7.18 (2H, d), 7.40 (1H, dd), 8.27 (1H, dd).

Example B238 2-(4-Butylbenzyl)-4-methoxypyridine

[1028]

[1029] The title compound was obtained in the same manner as in ExampleB211 using 2-chloro-4-methoxypyridine.

[1030]¹H-NMR (CDCl₃) δ (ppm): 0.91 (3H, t), 1.31-1.37 (2H, m), 1.53-1.59(2H, m), 2.57 (2H, t), 3.78 (3H, s), 4.06 (2H, s), 6.61-6.65 (2H, m),7.11 (2H, d), 7.17 (2H, d), 8.36 (1H, d).

Example B239 2-(4-Butylbenzyl)-4-chloropyridine

[1031]

[1032] Phosphorus oxychloride (57.0 μl, 0.612 mmol) was added to anice-cooled solution of the compound of Example B238 (52.0 mg, 0.204mmol) in dimethylformamide (1 ml), and this reaction mixture was stirredat 100° C. for 8 hours. The reaction mixture was allowed to cool, pouredon ice, and warmed to room temperature. After ethyl acetate was added,the mixture was washed with a saturated aqueous sodium hydrogencarbonatesolution and saturated brine, then concentrated under reduced pressure.The residue was purified by silica gel column chromatography to give thetitle compound (2.29 mg).

[1033]¹H-NMR (CDCl₃) δ (ppm): 0.92 (3H, t), 1.31-1.38 (2H, m), 1.53-1.61(2H, m), 2.59 (2H, t), 4.10 (2H, s), 7.12-18 (6H, m), 8.44 (1H, d).

Example B240 2-Chloro-3-methoxypyridine

[1034]

[1035] The title compound was obtained in the same manner as in ExampleB231 using 2-chloro-3-hydroxypyridine.

[1036]¹H-NMR (CDCl₃) δ (ppm): 3.93 (3H, s), 7.21-7.22 (2H, m), 7.99-8.01(1H, m).

Example B241 2-Chloro-3,4-dimethoxypyridine

[1037]

[1038] A solution of diisopropylamine (84.0 μl, 0.599 mmol) and thecompound of Example B240 (860 mg, 5.99 mmol) in tetrahydrofuran (4 ml)was added to a solution of 1.06 M phenyllithium cyclopentane-diethylether solution in tetrahydrofuran (11 ml) cooled to −78° C. undernitrogen atmosphere. This reaction mixture was stirred at −40° C. for 1hour, then at −18° C. for another 20 minutes. The reaction mixture wascooled again to −78° C., trimethoxyborate (2.04 ml, 18.0 mmol) was addeddropwise thereto, and the resulting mixture was stirred at 0° C. for 20minutes. At that temperature, aqueous ammonia (29%, 30 ml), ammoniumchloride (4.5 g,), and an aqueous hydrogen peroxide solution (30%, 12ml) were added in this order, and the mixture was stirred at roomtemperature for 2 hours. Saturated sodium thiosulfate, acetic acid andethyl acetate were added, and the mixture was washed with saturatedbrine. The ethyl acetate layer obtained upon filtration through silicagel was concentrated under reduced pressure. The resulting residue wastreated in the same manner as in Example B231 to obtain the titlecompound (31.3 mg).

[1039]¹H-NMR (CDCl₃) δ (ppm): 3.89 (3H, s), 3.94 (3H, s), 6.82 (1H, d),8.05 (1H, d).

Example B242 2-(4-Butylbenzyl)-3,4-dimethoxypyridine

[1040]

[1041] The title compound was obtained in the same manner as in ExampleB206 using the compound of Example B241.

[1042]¹H-NMR (CDCl₃) δ (ppm): 0.90 (3H, t), 1.26-1.35 (2H, m), 1.53-1.57(2H, m), 2.54 (2H, t), 3.70 (3H, s), 3.89 (3H, s), 4.12 (2H, s), 6.72(1H, d), 7.06 (2H, d), 7.21 (2H, d), 8.20 (1H, d).

Example B243 2,4-Di-(4-Butylbenzyl)-3-methoxypyridine

[1043]

[1044] A solution of the compound of Example B240 (436 mg, 3.04 mmol) indiethyl ether (2 ml) was added to a solution of 1.43 M t-butyllithiumn-pentane solution (2.76 ml, 3.95 mmol) in diethyl ether (5 ml) cooledto −78° C. under nitrogen atmosphere, and this reaction mixture wasstirred at that temperature for 30 minutes. A solution oftetramethylethylenediamine (688 μl, 4.56 mmol) and hexachloroethane (719mg, 3.04 mmol) in diethyl ether (3 ml) was further added and thereaction mixture was stirred at that temperature for 1 hour. Afterwarming gradually to room temperature, ethyl acetate was added, and themixture was washed with saturated brine. The ethyl acetate layerobtained upon filtration through silica gel was concentrated underreduced pressure. The resulting residue was treated in the same manneras in Example B206 to obtain the title compound (10.1 mg).

[1045]¹H-NMR (CDCl₃) δ (ppm): 0.89-0.94 (6H, m), 1.31-1.37 (4H, m),1.52-1.62 (4H, m), 2.53-2.59 (4H, m), 3.74 (3H, s), 4.07 (2H, s), 4.13(2H, s), 6.84 (1H, d), 6.98 (1H, d), 7.04-7.22 (8H, m).

Example B244 2-(4-Bromo-2-fluorobenzyl)-3-(methoxymethoxy)pyridine

[1046]

[1047] A solution of the compound of Example B228 (422 mg, 1.94 mmol) intetrahydrofuran (3 ml) was added to a solution of 2.47 M n-butyllithiumn-hexane solution (862 μl, 2.13 mmol) in tetrahydrofuran (3 ml) cooledto −78° C. under nitrogen atmosphere, and this reaction mixture wasstirred at that temperature for 1 hour. After copper(I) bromide (139 mg,0.968 mmol) was added, the reaction mixture was stirred at 0° C. for 1hour and cooled again to −78° C. Next, 4-bromo-2-fluorobenzyl bromide(259 mg, 0.968 mmol) was added, and the resulting mixture was stirred at0° C. for 1 hour. Tetramethylethylenediamine (584 μl, 3.88 mmol) wasfurther added, and the resulting reaction mixture was stirred at thattemperature for 1 hour. After diethyl ether and an aqueous ammoniasolution were added to the reaction mixture, the organic layer waswashed with saturated brine and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to give thetitle compound (81.0 mg).

[1048]¹H-NMR (CDCl₃) δ (ppm): 3.38 (3H, s), 4.17 (2H, s), 5.18 (2H, s),7.04 (1H, t), 7.11-7.22 (3H, m), 7.38 (1H, dd), 8.19 (1H, dd).

Example B245 2-(4-Bromo-2-fluorobenzyl)-3-pyridinol

[1049]

[1050] Trifluoroacetic acid (1 ml) was added to the compound of ExampleB244 (134 mg, 0.411 mmol) in methylene chloride (4 ml), and thisreaction mixture was stirred at room temperature overnight. Afterneutralizing the mixture with saturated aqueous sodiumhydrogencarbonate, ethyl acetate was added. The ethyl acetate layer waswashed with saturated brine and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to give thetitle compound (97.5 mg).

[1051]¹H-NMR (CDCl₃) δ (ppm): 4.17 (2H, s), 7.10-7.24 (5H, m), 8.15 (1H,t).

[1052] The proton of the phenolic hydroxyl group was not observed in theNMR spectrum.

Example B246 2-(4-Bromo-2-fluorobenzyl)-3-methoxypyridine

[1053]

[1054] Potassium carbonate (38.7 mg, 0.280 mmol) and methyl iodide (10.5μl, 0.168 mmol) were added to a solution of the compound of Example B245(15.8 mg, 0.0560 mmol) in dimethylformamide (1 ml), and this reactionmixture was stirred at room temperature for 2 hours. After ethyl acetatewas added, the reaction mixture was washed with saturated brine andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound (14.0 mg).

[1055]¹H-NMR (CDCl₃) δ (ppm): 3.82 (3H, s), 4.15 (2H, s), 7.03 (1H, t),7.12-7.22 (4H, m), 8.13 (1H, dd).

[1056] The following compounds of Example B were synthesized in the samemanner as in Example B246, and purification was performed by LC-MS[eluent: an acetonitrile solution containing 0.1% trifluoroacetic acid:an aqueous solution containing 0.1% trifluoroacetic acid=1:99 to100:0/20-minute cycle, flow rate: 20 ml/minute, column: YMC CombiprepODS-AM, 20 mm Φ×50 mm (long)].

Example B247 2-(4-Bromo-2-fluorobenzyl)-3-ethoxypyridine

[1057]

[1058] MS m/z (ESI: MH⁺): 310.0.

Example B248 2-(4-Bromo-2-fluorobenzyl)-3-propoxypyridine

[1059]

[1060] MS m/z (ESI: MH⁺): 324.0.

Example B249 2-(4-Bromo-2-fluorobenzyl)-3-butoxypyridine

[1061]

[1062] MS m/z (ESI: MH⁺): 338.1.

Example B250 2-(4-Bromo-2-fluorobenzyl)-3-(pentyloxy)pyridine

[1063]

[1064] MS m/z (ESI: MH⁺): 352.1

Example B251 2-(4-Bromo-2-fluorobenzyl)-3-(hexyloxy)pyridine

[1065]

[1066] MS m/z (ESI: MH⁺) 366.0

Example B252 2-(4-Bromo-2-fluorobenzyl)-3-(2-fluoroethoxy)pyridine

[1067]

[1068] MS m/z (ESI: MH⁺) 328.0

Example B253 2-(4-Bromo-2-fluorobenzyl)-3-(3-fluoropropoxy)pyridine

[1069]

[1070] MS m/z (ESI: MH⁺) 342.0

Example B254 2-(4-Bromo-2-fluorobenzyl)-3-isopropoxypyridine

[1071]

[1072] MS m/z (ESI: MH⁺): 324.0.

Example B2552-(4-Bromo-2-fluorobenzyl)-3-(2,2,2-trifluoroethoxy)pyridine

[1073]

[1074] MS m/z (ESI: MH⁺): 364.0.

Example B2562-(4-Bromo-2-fluorobenzyl)-3-(3,3,3-trifluoropropoxy)pyridine

[1075]

[1076] MS m/z (ESI: MH⁺): 378.0.

Example B257

[1077] Compounds were evaluated using the S. cerevisiae reporter systemof Example A2. The lowest concentration at which cephalosporinaseactivity in the cell wall fraction became 50% or less compared to thatobtained where the compound was not treated, was defined to be the IC50value. Effects of the representative compounds are shown in Table 1.TABLE 1 IC50 Compound (μg/ml) 1- (4-butylbenzyl)isoquinoline (ExampleB2) 0.39 N1-{3-[4-(1-isoquinolylmethyl)phenyl]-2-propynyl} 6.25acetamide (Example B60)N1-{3-[4-(1-isoquinolylmethyl)phenyl]propyl}-N1-methyl 50 acetamide(Example B73) 5-butyl-2-(1-isoquiriolylmethyl)phenol (Example B85) 0.204-(4-butylbenzyl)thieno[3,2-c]pyridine (Example B187) 0.787-(4-butylbenzyl)thieno[2,3-c]pyridine (Example B195) 0.392-(4-butylbenzyl)-3-methoxypyridine (Example B231) 0.782-(4-butylbenzyl)-3,4-dimethoxypyridine (Example B242) 0.78

INDUSTRIAL APPLICABILITY

[1078] The present invention revealed genes encoding the proteinsparticipating in the transport process of the GPI-anchored proteins tothe cell wall. Furthermore, this invention discloses a method ofscreening for compounds that inhibit the activity of these proteins, andalso discloses representative compounds having the inhibitory activity.

[1079] Using novel compounds, the present invention showed thatantifungal agents having a novel mechanism of inhibiting the transportprocess of the GPI-anchored proteins to the cell wall can be provided.

1 69 1 1497 DNA Saccharomyces cerevisiae CDS (1)..(1497) GWT1 1 atg gcaaca gta cat cag aag aat atg tcg act tta aaa cag aga aaa 48 Met Ala ThrVal His Gln Lys Asn Met Ser Thr Leu Lys Gln Arg Lys 1 5 10 15 gag gacttt gtg aca ggg ctc aat ggc ggt tct ata aca gaa att aac 96 Glu Asp PheVal Thr Gly Leu Asn Gly Gly Ser Ile Thr Glu Ile Asn 20 25 30 gca gtg acatca att gct ttg gta act tac ata tca tgg aac tta ttg 144 Ala Val Thr SerIle Ala Leu Val Thr Tyr Ile Ser Trp Asn Leu Leu 35 40 45 aaa aat tcc aacctt atg cct cct ggc att tcc agc gtg caa tac ata 192 Lys Asn Ser Asn LeuMet Pro Pro Gly Ile Ser Ser Val Gln Tyr Ile 50 55 60 att gat ttt gca ttgaac tgg gtt gct ttg ctt cta tct att act att 240 Ile Asp Phe Ala Leu AsnTrp Val Ala Leu Leu Leu Ser Ile Thr Ile 65 70 75 80 tat gct agt gaa ccatac ctt cta aac acg cta ata ctg tta cct tgt 288 Tyr Ala Ser Glu Pro TyrLeu Leu Asn Thr Leu Ile Leu Leu Pro Cys 85 90 95 ttg ctc gca ttc ata tatgga aaa ttt act agc tcg agt aaa cct tct 336 Leu Leu Ala Phe Ile Tyr GlyLys Phe Thr Ser Ser Ser Lys Pro Ser 100 105 110 aat cca ata tac aat aaaaaa aaa atg att aca cag cgg ttc caa cta 384 Asn Pro Ile Tyr Asn Lys LysLys Met Ile Thr Gln Arg Phe Gln Leu 115 120 125 gaa aaa aag ccg tat attact gcg tat cgt ggt ggg atg ctt att ctg 432 Glu Lys Lys Pro Tyr Ile ThrAla Tyr Arg Gly Gly Met Leu Ile Leu 130 135 140 act gct att gcc atc ttggct gta gat ttt cca att ttc cca agg agg 480 Thr Ala Ile Ala Ile Leu AlaVal Asp Phe Pro Ile Phe Pro Arg Arg 145 150 155 160 ttt gcc aag gtg gaaact tgg ggg aca tcc ctg atg gat ctt ggt gta 528 Phe Ala Lys Val Glu ThrTrp Gly Thr Ser Leu Met Asp Leu Gly Val 165 170 175 gga tca ttc gtt ttcagt aac ggt att gtt tct tct agg gca ctg ttg 576 Gly Ser Phe Val Phe SerAsn Gly Ile Val Ser Ser Arg Ala Leu Leu 180 185 190 aaa aac cta agc ttgaag agt aaa ccc agc ttc tta aaa aat gca ttt 624 Lys Asn Leu Ser Leu LysSer Lys Pro Ser Phe Leu Lys Asn Ala Phe 195 200 205 aat gcc tta aaa tcagga gga act cta ttg ttc cta gga ttg ctg agg 672 Asn Ala Leu Lys Ser GlyGly Thr Leu Leu Phe Leu Gly Leu Leu Arg 210 215 220 ttg ttt ttt gta aaaaat ttg gaa tat caa gaa cat gtc aca gaa tat 720 Leu Phe Phe Val Lys AsnLeu Glu Tyr Gln Glu His Val Thr Glu Tyr 225 230 235 240 ggg gtt cat tggaat ttt ttt atc acc cta tca ttg ttg cca ctt gta 768 Gly Val His Trp AsnPhe Phe Ile Thr Leu Ser Leu Leu Pro Leu Val 245 250 255 ttg acc ttt attgat ccc gtc aca aga atg gtt cca cgc tgc tca att 816 Leu Thr Phe Ile AspPro Val Thr Arg Met Val Pro Arg Cys Ser Ile 260 265 270 gca ata ttc atttca tgc att tat gaa tgg cta ctt tta aag gac gat 864 Ala Ile Phe Ile SerCys Ile Tyr Glu Trp Leu Leu Leu Lys Asp Asp 275 280 285 cgc act tta aacttt tta att ttg gct gat aga aat tgt ttc ttc agt 912 Arg Thr Leu Asn PheLeu Ile Leu Ala Asp Arg Asn Cys Phe Phe Ser 290 295 300 gct aat aga gaaggc atc ttc tca ttt cta ggt tat tgc tcg att ttt 960 Ala Asn Arg Glu GlyIle Phe Ser Phe Leu Gly Tyr Cys Ser Ile Phe 305 310 315 320 ctt tgg ggccaa aac acg gga ttt tac ttg ttg gga aat aaa cca act 1008 Leu Trp Gly GlnAsn Thr Gly Phe Tyr Leu Leu Gly Asn Lys Pro Thr 325 330 335 tta aac aatctt tat aag cct tct acg caa gac gta gtt gca gca tca 1056 Leu Asn Asn LeuTyr Lys Pro Ser Thr Gln Asp Val Val Ala Ala Ser 340 345 350 aag aag tcttcg act tgg gac tat tgg act tca gta acc cca tta agt 1104 Lys Lys Ser SerThr Trp Asp Tyr Trp Thr Ser Val Thr Pro Leu Ser 355 360 365 ggc ctc tgtata tgg agt aca att ttt ctt gtt atc agc cag ttg gtt 1152 Gly Leu Cys IleTrp Ser Thr Ile Phe Leu Val Ile Ser Gln Leu Val 370 375 380 ttt caa taccat cct tat agt gtt tca aga agg ttt gct aac tta cca 1200 Phe Gln Tyr HisPro Tyr Ser Val Ser Arg Arg Phe Ala Asn Leu Pro 385 390 395 400 tat actttg tgg gtc att act tat aat tta cta ttt ttg act ggg tac 1248 Tyr Thr LeuTrp Val Ile Thr Tyr Asn Leu Leu Phe Leu Thr Gly Tyr 405 410 415 tgc ttgact gac aaa att ttc ggt aat tct tcg gaa tat tat aaa gtt 1296 Cys Leu ThrAsp Lys Ile Phe Gly Asn Ser Ser Glu Tyr Tyr Lys Val 420 425 430 gcc gaatgc ttg gaa tca atc aac tcc aat ggg ttg ttt tta ttt ttg 1344 Ala Glu CysLeu Glu Ser Ile Asn Ser Asn Gly Leu Phe Leu Phe Leu 435 440 445 ttg gcaaat gtc tct act ggt tta gtc aat atg tct atg gtc acg ata 1392 Leu Ala AsnVal Ser Thr Gly Leu Val Asn Met Ser Met Val Thr Ile 450 455 460 gat tcttca ccc tta aaa tca ttc ctg gtt ttg ttg gca tac tgc tca 1440 Asp Ser SerPro Leu Lys Ser Phe Leu Val Leu Leu Ala Tyr Cys Ser 465 470 475 480 ttcata gct gtc ata tcg gtt ttc ttg tat aga aaa aga ata ttc att 1488 Phe IleAla Val Ile Ser Val Phe Leu Tyr Arg Lys Arg Ile Phe Ile 485 490 495 aagcta taa 1497 Lys Leu 2 498 PRT Saccharomyces cerevisiae GWT1 2 Met AlaThr Val His Gln Lys Asn Met Ser Thr Leu Lys Gln Arg Lys 1 5 10 15 GluAsp Phe Val Thr Gly Leu Asn Gly Gly Ser Ile Thr Glu Ile Asn 20 25 30 AlaVal Thr Ser Ile Ala Leu Val Thr Tyr Ile Ser Trp Asn Leu Leu 35 40 45 LysAsn Ser Asn Leu Met Pro Pro Gly Ile Ser Ser Val Gln Tyr Ile 50 55 60 IleAsp Phe Ala Leu Asn Trp Val Ala Leu Leu Leu Ser Ile Thr Ile 65 70 75 80Tyr Ala Ser Glu Pro Tyr Leu Leu Asn Thr Leu Ile Leu Leu Pro Cys 85 90 95Leu Leu Ala Phe Ile Tyr Gly Lys Phe Thr Ser Ser Ser Lys Pro Ser 100 105110 Asn Pro Ile Tyr Asn Lys Lys Lys Met Ile Thr Gln Arg Phe Gln Leu 115120 125 Glu Lys Lys Pro Tyr Ile Thr Ala Tyr Arg Gly Gly Met Leu Ile Leu130 135 140 Thr Ala Ile Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro ArgArg 145 150 155 160 Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Leu Met AspLeu Gly Val 165 170 175 Gly Ser Phe Val Phe Ser Asn Gly Ile Val Ser SerArg Ala Leu Leu 180 185 190 Lys Asn Leu Ser Leu Lys Ser Lys Pro Ser PheLeu Lys Asn Ala Phe 195 200 205 Asn Ala Leu Lys Ser Gly Gly Thr Leu LeuPhe Leu Gly Leu Leu Arg 210 215 220 Leu Phe Phe Val Lys Asn Leu Glu TyrGln Glu His Val Thr Glu Tyr 225 230 235 240 Gly Val His Trp Asn Phe PheIle Thr Leu Ser Leu Leu Pro Leu Val 245 250 255 Leu Thr Phe Ile Asp ProVal Thr Arg Met Val Pro Arg Cys Ser Ile 260 265 270 Ala Ile Phe Ile SerCys Ile Tyr Glu Trp Leu Leu Leu Lys Asp Asp 275 280 285 Arg Thr Leu AsnPhe Leu Ile Leu Ala Asp Arg Asn Cys Phe Phe Ser 290 295 300 Ala Asn ArgGlu Gly Ile Phe Ser Phe Leu Gly Tyr Cys Ser Ile Phe 305 310 315 320 LeuTrp Gly Gln Asn Thr Gly Phe Tyr Leu Leu Gly Asn Lys Pro Thr 325 330 335Leu Asn Asn Leu Tyr Lys Pro Ser Thr Gln Asp Val Val Ala Ala Ser 340 345350 Lys Lys Ser Ser Thr Trp Asp Tyr Trp Thr Ser Val Thr Pro Leu Ser 355360 365 Gly Leu Cys Ile Trp Ser Thr Ile Phe Leu Val Ile Ser Gln Leu Val370 375 380 Phe Gln Tyr His Pro Tyr Ser Val Ser Arg Arg Phe Ala Asn LeuPro 385 390 395 400 Tyr Thr Leu Trp Val Ile Thr Tyr Asn Leu Leu Phe LeuThr Gly Tyr 405 410 415 Cys Leu Thr Asp Lys Ile Phe Gly Asn Ser Ser GluTyr Tyr Lys Val 420 425 430 Ala Glu Cys Leu Glu Ser Ile Asn Ser Asn GlyLeu Phe Leu Phe Leu 435 440 445 Leu Ala Asn Val Ser Thr Gly Leu Val AsnMet Ser Met Val Thr Ile 450 455 460 Asp Ser Ser Pro Leu Lys Ser Phe LeuVal Leu Leu Ala Tyr Cys Ser 465 470 475 480 Phe Ile Ala Val Ile Ser ValPhe Leu Tyr Arg Lys Arg Ile Phe Ile 485 490 495 Lys Leu 3 1458 DNACandida albicans CDS (1)..(1458) CaGWT1 3 atg tca tcg tct tta aaa caattg aaa gaa caa ttt gtc tca gat ttg 48 Met Ser Ser Ser Leu Lys Gln LeuLys Glu Gln Phe Val Ser Asp Leu 1 5 10 15 act ggt ggc aca att gaa gaaatt tat gct gta acc agt ata gca tta 96 Thr Gly Gly Thr Ile Glu Glu IleTyr Ala Val Thr Ser Ile Ala Leu 20 25 30 tca tct tat ttg tcc ttt aga ttgttg aaa aag tct ctt ggt gat tta 144 Ser Ser Tyr Leu Ser Phe Arg Leu LeuLys Lys Ser Leu Gly Asp Leu 35 40 45 gct ttg att tac gac tac att ctt aatgtg ttg aca att cta gca tcc 192 Ala Leu Ile Tyr Asp Tyr Ile Leu Asn ValLeu Thr Ile Leu Ala Ser 50 55 60 att act gtt tat agc aac agc cct tct tatttg cat tat ttt att gtt 240 Ile Thr Val Tyr Ser Asn Ser Pro Ser Tyr LeuHis Tyr Phe Ile Val 65 70 75 80 att cca tca tta gtt ata tat cta gtg aattac cat gtt gag aaa cca 288 Ile Pro Ser Leu Val Ile Tyr Leu Val Asn TyrHis Val Glu Lys Pro 85 90 95 tct tca ccc cat aga caa aat gat aca aaa gaagat aaa tcg gac gaa 336 Ser Ser Pro His Arg Gln Asn Asp Thr Lys Glu AspLys Ser Asp Glu 100 105 110 cta ttg ccg aga aaa caa ttt ata aca gcc tatcgt tct caa atg ttg 384 Leu Leu Pro Arg Lys Gln Phe Ile Thr Ala Tyr ArgSer Gln Met Leu 115 120 125 ata att act aat cta gct ata tta gct gtt gatttt cct att ttc cca 432 Ile Ile Thr Asn Leu Ala Ile Leu Ala Val Asp PhePro Ile Phe Pro 130 135 140 aga aga ttt gcc aaa gtg gaa aca tgg ggc acgtca atg atg gat tta 480 Arg Arg Phe Ala Lys Val Glu Thr Trp Gly Thr SerMet Met Asp Leu 145 150 155 160 gga gtt ggg tcg ttt gtg ttc tcc atg gggttg gct aat tct cga caa 528 Gly Val Gly Ser Phe Val Phe Ser Met Gly LeuAla Asn Ser Arg Gln 165 170 175 ttg atc aag aac cac acc gac aac tac aaattt agt tgg aag agt tat 576 Leu Ile Lys Asn His Thr Asp Asn Tyr Lys PheSer Trp Lys Ser Tyr 180 185 190 ttg aaa aca atc aag cag aac ttt atc aagtca gtg cct ata ctt gtt 624 Leu Lys Thr Ile Lys Gln Asn Phe Ile Lys SerVal Pro Ile Leu Val 195 200 205 tta gga gct att cgt ttt gtt agt gtt aagcaa ttg gac tat cag gaa 672 Leu Gly Ala Ile Arg Phe Val Ser Val Lys GlnLeu Asp Tyr Gln Glu 210 215 220 cac gaa aca gag tat gga atc cat tgg aatttt ttc ttc aca tta ggg 720 His Glu Thr Glu Tyr Gly Ile His Trp Asn PhePhe Phe Thr Leu Gly 225 230 235 240 ttc ttg cca att gta ttg gga ata ttagac ccg gtg ttg aat ttg gtt 768 Phe Leu Pro Ile Val Leu Gly Ile Leu AspPro Val Leu Asn Leu Val 245 250 255 cca cgc ttc ata ata gga att ggt atctca att gct tat gag gta gcg 816 Pro Arg Phe Ile Ile Gly Ile Gly Ile SerIle Ala Tyr Glu Val Ala 260 265 270 ttg aat aag act ggt ttg ttg aag ttcatt ttg agc agc gaa aac aga 864 Leu Asn Lys Thr Gly Leu Leu Lys Phe IleLeu Ser Ser Glu Asn Arg 275 280 285 ctt gaa tct ctc atc acc atg aat aaagaa ggt att ttt tcg ttt att 912 Leu Glu Ser Leu Ile Thr Met Asn Lys GluGly Ile Phe Ser Phe Ile 290 295 300 gga tat ctt tgt att ttt ata att ggtcag tct ttt ggg tca ttt gtt 960 Gly Tyr Leu Cys Ile Phe Ile Ile Gly GlnSer Phe Gly Ser Phe Val 305 310 315 320 tta aca ggc tac aaa aca aag aacaac tta ata acc att agc aaa att 1008 Leu Thr Gly Tyr Lys Thr Lys Asn AsnLeu Ile Thr Ile Ser Lys Ile 325 330 335 cgt att tca aaa aaa caa cac aagaaa gag ctg ctg ctg ttt ttc tca 1056 Arg Ile Ser Lys Lys Gln His Lys LysGlu Leu Leu Leu Phe Phe Ser 340 345 350 gtc gcc act act cag gga tta tatttg gca tgt atc ttc tat cac tta 1104 Val Ala Thr Thr Gln Gly Leu Tyr LeuAla Cys Ile Phe Tyr His Leu 355 360 365 gct ttc agt ttg ttc atc agc aactta tca ttc ttg caa cca att tca 1152 Ala Phe Ser Leu Phe Ile Ser Asn LeuSer Phe Leu Gln Pro Ile Ser 370 375 380 aga cga ttg gcc aat ttc ccc tacgtc atg tgg gtc gtt tcg tac aat 1200 Arg Arg Leu Ala Asn Phe Pro Tyr ValMet Trp Val Val Ser Tyr Asn 385 390 395 400 gct acg ttt tta tta tgt tatgac tta att gaa aaa ttt atc ccg ggg 1248 Ala Thr Phe Leu Leu Cys Tyr AspLeu Ile Glu Lys Phe Ile Pro Gly 405 410 415 aac ctt act tct act gta ttggac tct att aat aac aat ggt tta ttt 1296 Asn Leu Thr Ser Thr Val Leu AspSer Ile Asn Asn Asn Gly Leu Phe 420 425 430 atc ttc ttg gtc agc aat ttatta aca ggg ttt att aac atg tcc atc 1344 Ile Phe Leu Val Ser Asn Leu LeuThr Gly Phe Ile Asn Met Ser Ile 435 440 445 aac act ttg gaa act agc aataaa atg gca gtg att atc ttg att ggc 1392 Asn Thr Leu Glu Thr Ser Asn LysMet Ala Val Ile Ile Leu Ile Gly 450 455 460 tat agt ctt act tgg aca ttgctc gcc tta tat ttg gat aag agg aag 1440 Tyr Ser Leu Thr Trp Thr Leu LeuAla Leu Tyr Leu Asp Lys Arg Lys 465 470 475 480 atc tac atc aag ctt tag1458 Ile Tyr Ile Lys Leu 485 4 485 PRT Candida albicans CaGWT1 4 Met SerSer Ser Leu Lys Gln Leu Lys Glu Gln Phe Val Ser Asp Leu 1 5 10 15 ThrGly Gly Thr Ile Glu Glu Ile Tyr Ala Val Thr Ser Ile Ala Leu 20 25 30 SerSer Tyr Leu Ser Phe Arg Leu Leu Lys Lys Ser Leu Gly Asp Leu 35 40 45 AlaLeu Ile Tyr Asp Tyr Ile Leu Asn Val Leu Thr Ile Leu Ala Ser 50 55 60 IleThr Val Tyr Ser Asn Ser Pro Ser Tyr Leu His Tyr Phe Ile Val 65 70 75 80Ile Pro Ser Leu Val Ile Tyr Leu Val Asn Tyr His Val Glu Lys Pro 85 90 95Ser Ser Pro His Arg Gln Asn Asp Thr Lys Glu Asp Lys Ser Asp Glu 100 105110 Leu Leu Pro Arg Lys Gln Phe Ile Thr Ala Tyr Arg Ser Gln Met Leu 115120 125 Ile Ile Thr Asn Leu Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro130 135 140 Arg Arg Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Met Met AspLeu 145 150 155 160 Gly Val Gly Ser Phe Val Phe Ser Met Gly Leu Ala AsnSer Arg Gln 165 170 175 Leu Ile Lys Asn His Thr Asp Asn Tyr Lys Phe SerTrp Lys Ser Tyr 180 185 190 Leu Lys Thr Ile Lys Gln Asn Phe Ile Lys SerVal Pro Ile Leu Val 195 200 205 Leu Gly Ala Ile Arg Phe Val Ser Val LysGln Leu Asp Tyr Gln Glu 210 215 220 His Glu Thr Glu Tyr Gly Ile His TrpAsn Phe Phe Phe Thr Leu Gly 225 230 235 240 Phe Leu Pro Ile Val Leu GlyIle Leu Asp Pro Val Leu Asn Leu Val 245 250 255 Pro Arg Phe Ile Ile GlyIle Gly Ile Ser Ile Ala Tyr Glu Val Ala 260 265 270 Leu Asn Lys Thr GlyLeu Leu Lys Phe Ile Leu Ser Ser Glu Asn Arg 275 280 285 Leu Glu Ser LeuIle Thr Met Asn Lys Glu Gly Ile Phe Ser Phe Ile 290 295 300 Gly Tyr LeuCys Ile Phe Ile Ile Gly Gln Ser Phe Gly Ser Phe Val 305 310 315 320 LeuThr Gly Tyr Lys Thr Lys Asn Asn Leu Ile Thr Ile Ser Lys Ile 325 330 335Arg Ile Ser Lys Lys Gln His Lys Lys Glu Leu Leu Leu Phe Phe Ser 340 345350 Val Ala Thr Thr Gln Gly Leu Tyr Leu Ala Cys Ile Phe Tyr His Leu 355360 365 Ala Phe Ser Leu Phe Ile Ser Asn Leu Ser Phe Leu Gln Pro Ile Ser370 375 380 Arg Arg Leu Ala Asn Phe Pro Tyr Val Met Trp Val Val Ser TyrAsn 385 390 395 400 Ala Thr Phe Leu Leu Cys Tyr Asp Leu Ile Glu Lys PheIle Pro Gly 405 410 415 Asn Leu Thr Ser Thr Val Leu Asp Ser Ile Asn AsnAsn Gly Leu Phe 420 425 430 Ile Phe Leu Val Ser Asn Leu Leu Thr Gly PheIle Asn Met Ser Ile 435 440 445 Asn Thr Leu Glu Thr Ser Asn Lys Met AlaVal Ile Ile Leu Ile Gly 450 455 460 Tyr Ser Leu Thr Trp Thr Leu Leu AlaLeu Tyr Leu Asp Lys Arg Lys 465 470 475 480 Ile Tyr Ile Lys Leu 485 51458 DNA Candida albicans CDS (1)..(1458) CaGWT1 5 atg tca tcg tct ttaaaa caa ttg aaa gaa caa ttt gtc tca gat ttg 48 Met Ser Ser Ser Leu LysGln Leu Lys Glu Gln Phe Val Ser Asp Leu 1 5 10 15 act ggt ggc aca attgaa gaa att tat gct gta acc agt ata gca tta 96 Thr Gly Gly Thr Ile GluGlu Ile Tyr Ala Val Thr Ser Ile Ala Leu 20 25 30 tca tct tat ttg tcc tttaga ttg ttg aaa aag tct ctt ggt gat tta 144 Ser Ser Tyr Leu Ser Phe ArgLeu Leu Lys Lys Ser Leu Gly Asp Leu 35 40 45 gct ttg att tac gac tac attctt aat gtg ttg aca att cta gca tcc 192 Ala Leu Ile Tyr Asp Tyr Ile LeuAsn Val Leu Thr Ile Leu Ala Ser 50 55 60 att act gtt tat agc aac agc ccttct tat ttg cat tat ttt att gtt 240 Ile Thr Val Tyr Ser Asn Ser Pro SerTyr Leu His Tyr Phe Ile Val 65 70 75 80 att cca tca tta gtt ata tat ctagtg aat tac cat gtt gag aaa cca 288 Ile Pro Ser Leu Val Ile Tyr Leu ValAsn Tyr His Val Glu Lys Pro 85 90 95 tct tca ccc cat aga caa aat gat acaaaa gaa gat aaa tcg gac gaa 336 Ser Ser Pro His Arg Gln Asn Asp Thr LysGlu Asp Lys Ser Asp Glu 100 105 110 cta ttg ccg aga aaa caa ttt ata acagcc tat cgt tct caa atg ttg 384 Leu Leu Pro Arg Lys Gln Phe Ile Thr AlaTyr Arg Ser Gln Met Leu 115 120 125 ata att act aat cta gct ata tta gctgtt gat ttt cct att ttc cca 432 Ile Ile Thr Asn Leu Ala Ile Leu Ala ValAsp Phe Pro Ile Phe Pro 130 135 140 aga aga ttt gcc aaa gtg gaa aca tggggc acg tca atg atg gat tta 480 Arg Arg Phe Ala Lys Val Glu Thr Trp GlyThr Ser Met Met Asp Leu 145 150 155 160 gga gtt ggg tcg ttt gtg ttc tccatg ggg ttg gct aat tct cga caa 528 Gly Val Gly Ser Phe Val Phe Ser MetGly Leu Ala Asn Ser Arg Gln 165 170 175 ttg atc aag aac cac acc gac aattac aaa ttt agt tgg aag agt tat 576 Leu Ile Lys Asn His Thr Asp Asn TyrLys Phe Ser Trp Lys Ser Tyr 180 185 190 ttg aaa aca atc aag cag aac tttatc aag tca gtg cct ata ctt gtt 624 Leu Lys Thr Ile Lys Gln Asn Phe IleLys Ser Val Pro Ile Leu Val 195 200 205 tta gga gct att cgt ttt gtt agtgtt aag caa ttg gac tat cag gaa 672 Leu Gly Ala Ile Arg Phe Val Ser ValLys Gln Leu Asp Tyr Gln Glu 210 215 220 cac gaa aca gag tat gga atc cattgg aat ttt ttc ttc aca tta ggg 720 His Glu Thr Glu Tyr Gly Ile His TrpAsn Phe Phe Phe Thr Leu Gly 225 230 235 240 ttc ttg cca att gta ttg ggaata tta gac ccg gtg ttg aat ttg gtt 768 Phe Leu Pro Ile Val Leu Gly IleLeu Asp Pro Val Leu Asn Leu Val 245 250 255 cca cgc ttc ata ata gga attggt atc tca att ggt tat gag gta gcg 816 Pro Arg Phe Ile Ile Gly Ile GlyIle Ser Ile Gly Tyr Glu Val Ala 260 265 270 ttg aat aag act ggt ttg ttgaag ttc att ttg agc agc gaa aac aga 864 Leu Asn Lys Thr Gly Leu Leu LysPhe Ile Leu Ser Ser Glu Asn Arg 275 280 285 ctt gaa tct ctc atc gcc atgaat aaa gaa ggt att ttt tcg ttt att 912 Leu Glu Ser Leu Ile Ala Met AsnLys Glu Gly Ile Phe Ser Phe Ile 290 295 300 gga tat ctt tgt att ttt ataatt ggt cag tct ttt ggg tca ttt gtt 960 Gly Tyr Leu Cys Ile Phe Ile IleGly Gln Ser Phe Gly Ser Phe Val 305 310 315 320 tta aca ggc tac aaa acaaag aac aac tta ata acc att agc aaa att 1008 Leu Thr Gly Tyr Lys Thr LysAsn Asn Leu Ile Thr Ile Ser Lys Ile 325 330 335 cgt att tca aaa aaa caacac aag aaa gag ctg ctg ctg ttt ttc tca 1056 Arg Ile Ser Lys Lys Gln HisLys Lys Glu Leu Leu Leu Phe Phe Ser 340 345 350 gtc gcc act act cag ggatta tat ttg gca tgt atc ttc tat cac tta 1104 Val Ala Thr Thr Gln Gly LeuTyr Leu Ala Cys Ile Phe Tyr His Leu 355 360 365 gct ttc agt ttg ttc atcagc aac tta tca ttc ttg caa cca att tca 1152 Ala Phe Ser Leu Phe Ile SerAsn Leu Ser Phe Leu Gln Pro Ile Ser 370 375 380 aga cga ttg gcc aat ttcccc tac gtc atg tgg gtc gtt tcg tac aat 1200 Arg Arg Leu Ala Asn Phe ProTyr Val Met Trp Val Val Ser Tyr Asn 385 390 395 400 gct acg ttt tta ttatgt tat gac tta att gaa aaa ttt atc ccg ggg 1248 Ala Thr Phe Leu Leu CysTyr Asp Leu Ile Glu Lys Phe Ile Pro Gly 405 410 415 aac ctt act tct actgta ttg gac tct att aat aac aat ggt tta ttt 1296 Asn Leu Thr Ser Thr ValLeu Asp Ser Ile Asn Asn Asn Gly Leu Phe 420 425 430 atc ttc ttg gtc agcaat tta tta aca ggg ttt att aac atg tcc atc 1344 Ile Phe Leu Val Ser AsnLeu Leu Thr Gly Phe Ile Asn Met Ser Ile 435 440 445 aac act ttg gaa actagc aat aaa atg gca gtg att atc ttg att ggc 1392 Asn Thr Leu Glu Thr SerAsn Lys Met Ala Val Ile Ile Leu Ile Gly 450 455 460 tat agt ctt act tggaca ttg ctc gcc tta tat ttg gat aag agg aag 1440 Tyr Ser Leu Thr Trp ThrLeu Leu Ala Leu Tyr Leu Asp Lys Arg Lys 465 470 475 480 atc tac atc aagctt tag 1458 Ile Tyr Ile Lys Leu 485 6 485 PRT Candida albicans CaGWT1 6Met Ser Ser Ser Leu Lys Gln Leu Lys Glu Gln Phe Val Ser Asp Leu 1 5 1015 Thr Gly Gly Thr Ile Glu Glu Ile Tyr Ala Val Thr Ser Ile Ala Leu 20 2530 Ser Ser Tyr Leu Ser Phe Arg Leu Leu Lys Lys Ser Leu Gly Asp Leu 35 4045 Ala Leu Ile Tyr Asp Tyr Ile Leu Asn Val Leu Thr Ile Leu Ala Ser 50 5560 Ile Thr Val Tyr Ser Asn Ser Pro Ser Tyr Leu His Tyr Phe Ile Val 65 7075 80 Ile Pro Ser Leu Val Ile Tyr Leu Val Asn Tyr His Val Glu Lys Pro 8590 95 Ser Ser Pro His Arg Gln Asn Asp Thr Lys Glu Asp Lys Ser Asp Glu100 105 110 Leu Leu Pro Arg Lys Gln Phe Ile Thr Ala Tyr Arg Ser Gln MetLeu 115 120 125 Ile Ile Thr Asn Leu Ala Ile Leu Ala Val Asp Phe Pro IlePhe Pro 130 135 140 Arg Arg Phe Ala Lys Val Glu Thr Trp Gly Thr Ser MetMet Asp Leu 145 150 155 160 Gly Val Gly Ser Phe Val Phe Ser Met Gly LeuAla Asn Ser Arg Gln 165 170 175 Leu Ile Lys Asn His Thr Asp Asn Tyr LysPhe Ser Trp Lys Ser Tyr 180 185 190 Leu Lys Thr Ile Lys Gln Asn Phe IleLys Ser Val Pro Ile Leu Val 195 200 205 Leu Gly Ala Ile Arg Phe Val SerVal Lys Gln Leu Asp Tyr Gln Glu 210 215 220 His Glu Thr Glu Tyr Gly IleHis Trp Asn Phe Phe Phe Thr Leu Gly 225 230 235 240 Phe Leu Pro Ile ValLeu Gly Ile Leu Asp Pro Val Leu Asn Leu Val 245 250 255 Pro Arg Phe IleIle Gly Ile Gly Ile Ser Ile Gly Tyr Glu Val Ala 260 265 270 Leu Asn LysThr Gly Leu Leu Lys Phe Ile Leu Ser Ser Glu Asn Arg 275 280 285 Leu GluSer Leu Ile Ala Met Asn Lys Glu Gly Ile Phe Ser Phe Ile 290 295 300 GlyTyr Leu Cys Ile Phe Ile Ile Gly Gln Ser Phe Gly Ser Phe Val 305 310 315320 Leu Thr Gly Tyr Lys Thr Lys Asn Asn Leu Ile Thr Ile Ser Lys Ile 325330 335 Arg Ile Ser Lys Lys Gln His Lys Lys Glu Leu Leu Leu Phe Phe Ser340 345 350 Val Ala Thr Thr Gln Gly Leu Tyr Leu Ala Cys Ile Phe Tyr HisLeu 355 360 365 Ala Phe Ser Leu Phe Ile Ser Asn Leu Ser Phe Leu Gln ProIle Ser 370 375 380 Arg Arg Leu Ala Asn Phe Pro Tyr Val Met Trp Val ValSer Tyr Asn 385 390 395 400 Ala Thr Phe Leu Leu Cys Tyr Asp Leu Ile GluLys Phe Ile Pro Gly 405 410 415 Asn Leu Thr Ser Thr Val Leu Asp Ser IleAsn Asn Asn Gly Leu Phe 420 425 430 Ile Phe Leu Val Ser Asn Leu Leu ThrGly Phe Ile Asn Met Ser Ile 435 440 445 Asn Thr Leu Glu Thr Ser Asn LysMet Ala Val Ile Ile Leu Ile Gly 450 455 460 Tyr Ser Leu Thr Trp Thr LeuLeu Ala Leu Tyr Leu Asp Lys Arg Lys 465 470 475 480 Ile Tyr Ile Lys Leu485 7 1458 DNA Candida albicans homologue of S. cerevisiae GWT1 7atgtcatcgt ctttaaaaca attgaaagaa caatttgtct cagatttgac tggtggcaca 60attgaagaaa tttatgctgt aaccagtata gcattatcat cttatttgtc ctttagattg 120ttgaaaaagt ctcttggtga tttagctttg atttacgact acattcttaa tgtgttgaca 180attctagcat ccattactgt ttatagcaac agcccttctt atttgcatta ttttattgtt 240attccatcat tagttatata tctagtgaat taccatgttg agaaaccatc ttcaccccat 300agacaaaatg atacaaaaga agataaatcg gacgaactat tgccgagaaa acaatttata 360acagcctatc gttctcaaat gttgataatt actaatctag ctatattagc tgttgatttt 420cctattttcc caagaagatt tgccaaagtg gaaacatggg gcacgtcaat gatggattta 480ggggttgggt cgtttgtgtt ctccatgggg ttggctaatt ctcgacaatt gatcaagaac 540cacaccgaca actacaaatt tagttggaag agttatttga aaacaatcaa gcagaacttt 600atcaagtcag tgcctatact tgttttagga gctattcgtt ttgttagtgt taagcaattg 660gactatcagg aacacgaaac agagtatgga atccattgga attttttctt cacattaggg 720ttcttgccaa ttgtattggg aatattagac ccggtgttga atttggttcc acgcttcata 780ataggaattg gtatctcaat tggttatgag gtagcgttga ataagactgg tttgttgaag 840ttcattttga gcagcgaaaa cagacttgaa tctctcatcg ccatgaataa agaaggtatt 900ttttcgttta ttggatatct ttgtattttt ataattggtc agtcttttgg gtcatttgtt 960ttaacaggct acaaaacaaa gaacaactta ataaccatta gcaaaattcg tatttcaaaa 1020aaacaacaca agaaagagct gctgctgttt ttctcagtcg ccactactca gggattatat 1080ttggcatgta tcttctatca cttagctttc agtttgttca tcagcaactt atcattcttg 1140caaccaattt caagacgatt ggccaatttc ccctacgtca tgtgggtcgt ttcgtacaat 1200gctacgtttt tattatgtta tgacttaatt gaaaaattta tcccggggaa ccttacttct 1260actgtattgg attctattaa taacaatggt ttatttatct tcttggtcag caatttatta 1320acagggttta ttaacatgtc catcaacact ttggaaacta gcaataaaat ggcagtgatt 1380atcttgattg gctatagtct tacttggaca ttgctcgcct tatatttgga taagaggaag 1440atctacatca agctttag 1458 8 33 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer 8 gcagtcgact cgatgaggtctttgctaatc ttg 33 9 33 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 9 gcagaattcg acaccacaac cttgaacgta ttg33 10 33 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 10 cccgaattca ctgacggtca aatccaagct act 33 11 32DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 11 ggaagctttt ataacaacat agcggcagca gc 32 12 49DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 12 cccgcggccg cttgatagta agcttgcttg ggccgcatcatgtaattag 49 13 33 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 13 cccggtacca aattaaagcc ttcgagcctc cca33 14 33 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 14 cccggatcct gtttgcagca tgagacttgc ata 33 15 45DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 15 cccgcggccg ccccttccaa ttcgaaaacc ttccccagagcagcc 45 16 32 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 16 ggttcgaagc cgcaaaaaca gaacaacaaa tt32 17 32 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 17 ggtctagatt gcagtttttc aagaatgcgc ca 32 18 33DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 18 gggtctagaa ctgacggtca aatccaagct act 33 19 32DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 19 ggaagctttt ataacaacat agcggcagca gc 32 20 17PRT Artificial Sequence Description of Artificial SequenceA1s1psynthetic peptide 20 Cys Phe Thr Ala Gly Thr Asn Thr Val Phe Asn Asp GlyAsp Lys Asp 1 5 10 15 Ile 21 27 DNA Artificial Sequence Description ofArtificial SequencePCR amplification primer 21 aaactgttca ctgaacaaccaaatctc 27 22 27 DNA Artificial Sequence Description of ArtificialSequencePCR amplification primer 22 caactgtacc atttgttaga catcact 27 2330 DNA Artificial Sequence Description of Artificial SequencePCRamplification primer 23 aaacagctgg gatcgcaata agaagacacg 30 24 29 DNAArtificial Sequence Description of Artificial SequencePCR amplificationprimer 24 aaacagctga tggaaatgtg gatggtgtg 29 25 60 DNA ArtificialSequence Description of Artificial SequencePCR amplification primer 25atggcaacag tacatcagga gaatatgtcg actttaaaac cggatccccg tcgtttaaac 60 2660 DNA Artificial Sequence Description of Artificial SequencePCRamplification primer 26 ttatagctta atgaatattc tttttctata caagaaaaccgaattcgagc tcgtttaaac 60 27 1380 DNA Schizosaccharomyces pombe CDS(1)..(1380) homologue of S. cerevisiae GWT1 27 atg tca tac aaa ttg gaaaaa gaa gca ttt gtc tca aac ctg acg ggt 48 Met Ser Tyr Lys Leu Glu LysGlu Ala Phe Val Ser Asn Leu Thr Gly 1 5 10 15 tca agt tcc att gag acatgt ggc ttg tta tta ata gga att gct tgc 96 Ser Ser Ser Ile Glu Thr CysGly Leu Leu Leu Ile Gly Ile Ala Cys 20 25 30 aac gtt ttg tgg gta aac atgact gcg aga aac atc tta ccc aaa ggg 144 Asn Val Leu Trp Val Asn Met ThrAla Arg Asn Ile Leu Pro Lys Gly 35 40 45 aat ctt ggg ttt ctt gtt gag tttttc atc ttt tgc tta att cca tta 192 Asn Leu Gly Phe Leu Val Glu Phe PheIle Phe Cys Leu Ile Pro Leu 50 55 60 ttt gtc att tac gtt tca tcg aaa gttggc gtt ttc act ctt tgc ata 240 Phe Val Ile Tyr Val Ser Ser Lys Val GlyVal Phe Thr Leu Cys Ile 65 70 75 80 gcc tct ttt ttg cct tcc ttc gtc cttcat gtt ata agt cca att aat 288 Ala Ser Phe Leu Pro Ser Phe Val Leu HisVal Ile Ser Pro Ile Asn 85 90 95 tgg gat gtg ctg aga aga aaa cct ggt tgttgt ctt act aaa aaa aat 336 Trp Asp Val Leu Arg Arg Lys Pro Gly Cys CysLeu Thr Lys Lys Asn 100 105 110 gaa aat act ttt gat cga cga att gct ggagtc aca ttt tat cgt tct 384 Glu Asn Thr Phe Asp Arg Arg Ile Ala Gly ValThr Phe Tyr Arg Ser 115 120 125 caa atg atg ttg gtt act gtc act tgc atcctg gcc gtt gac ttt acc 432 Gln Met Met Leu Val Thr Val Thr Cys Ile LeuAla Val Asp Phe Thr 130 135 140 ctt ttc ccg agg aga tat gcc aaa gtt gaaacc tgg gga aca tca ctg 480 Leu Phe Pro Arg Arg Tyr Ala Lys Val Glu ThrTrp Gly Thr Ser Leu 145 150 155 160 atg gat ctt ggt gtt gga tct ttc atgttt tct tca ggt act gtg gct 528 Met Asp Leu Gly Val Gly Ser Phe Met PheSer Ser Gly Thr Val Ala 165 170 175 gga cgg aaa aat gac att aaa aaa ccaaat gcg ttt aaa aat gta ttg 576 Gly Arg Lys Asn Asp Ile Lys Lys Pro AsnAla Phe Lys Asn Val Leu 180 185 190 tgg aat tct ttc atc ctt ttg att ttagga ttt gcg cgc atg ttt tta 624 Trp Asn Ser Phe Ile Leu Leu Ile Leu GlyPhe Ala Arg Met Phe Leu 195 200 205 acg aaa agc atc aat tac caa gaa catgta agc gaa tat ggc atg cat 672 Thr Lys Ser Ile Asn Tyr Gln Glu His ValSer Glu Tyr Gly Met His 210 215 220 tgg aac ttt ttt ttc acc cta ggt ttcatg gct ctt ggc gta ttt ttt 720 Trp Asn Phe Phe Phe Thr Leu Gly Phe MetAla Leu Gly Val Phe Phe 225 230 235 240 ttt cgt cgt tct tta aaa aaa gtctcc tat ttt aat tta gca acc ttc 768 Phe Arg Arg Ser Leu Lys Lys Val SerTyr Phe Asn Leu Ala Thr Phe 245 250 255 att act ctt ctt cat cat tgt ttgctt gtt tta acc cct ttc caa aaa 816 Ile Thr Leu Leu His His Cys Leu LeuVal Leu Thr Pro Phe Gln Lys 260 265 270 tgg gca cta tcc gcc ccc aga acaaat att ttg gct cag aat aga gag 864 Trp Ala Leu Ser Ala Pro Arg Thr AsnIle Leu Ala Gln Asn Arg Glu 275 280 285 ggt att gct tct ctt ccc gga tacatt gct att tac ttt tat gga atg 912 Gly Ile Ala Ser Leu Pro Gly Tyr IleAla Ile Tyr Phe Tyr Gly Met 290 295 300 tat acc ggt agt gta gtt ttg gctgat cga cct cta atg tat act aga 960 Tyr Thr Gly Ser Val Val Leu Ala AspArg Pro Leu Met Tyr Thr Arg 305 310 315 320 gct gag tcg tgg aag cgc tttcaa cgt cta tta ttc ccg cta tgc att 1008 Ala Glu Ser Trp Lys Arg Phe GlnArg Leu Leu Phe Pro Leu Cys Ile 325 330 335 ttg tta gtg ttg tat ctt gtgtct aac ttt ttg tca gtt ggt gtt tct 1056 Leu Leu Val Leu Tyr Leu Val SerAsn Phe Leu Ser Val Gly Val Ser 340 345 350 cgc cga ctt gct aat acg ccttat gtt gcg aat gtt gcc ttt atc aat 1104 Arg Arg Leu Ala Asn Thr Pro TyrVal Ala Asn Val Ala Phe Ile Asn 355 360 365 atg ttt ttt ctt act ata tacata ctt att gat gcc tat tta ttc cca 1152 Met Phe Phe Leu Thr Ile Tyr IleLeu Ile Asp Ala Tyr Leu Phe Pro 370 375 380 tct tct gtg cca tat gga agtcgc gtc ccc aaa ctg ctt gaa gat gcc 1200 Ser Ser Val Pro Tyr Gly Ser ArgVal Pro Lys Leu Leu Glu Asp Ala 385 390 395 400 aat aat aat ggc ttg ttggtg ttt ttg att gct aac gtt tta aca gga 1248 Asn Asn Asn Gly Leu Leu ValPhe Leu Ile Ala Asn Val Leu Thr Gly 405 410 415 gta gtt aat tta tcg ttcgac acc ctt cat tct agc aat gca aaa ggc 1296 Val Val Asn Leu Ser Phe AspThr Leu His Ser Ser Asn Ala Lys Gly 420 425 430 ttg aca atc atg act atgtat ctt ttt att att tgc tat atg gca cat 1344 Leu Thr Ile Met Thr Met TyrLeu Phe Ile Ile Cys Tyr Met Ala His 435 440 445 tgg ctt gct caa cac ggaatt cgt ttt cgc ctt tag 1380 Trp Leu Ala Gln His Gly Ile Arg Phe Arg Leu450 455 460 28 459 PRT Schizosaccharomyces pombe homologue of S.cerevisiae GWT1 28 Met Ser Tyr Lys Leu Glu Lys Glu Ala Phe Val Ser AsnLeu Thr Gly 1 5 10 15 Ser Ser Ser Ile Glu Thr Cys Gly Leu Leu Leu IleGly Ile Ala Cys 20 25 30 Asn Val Leu Trp Val Asn Met Thr Ala Arg Asn IleLeu Pro Lys Gly 35 40 45 Asn Leu Gly Phe Leu Val Glu Phe Phe Ile Phe CysLeu Ile Pro Leu 50 55 60 Phe Val Ile Tyr Val Ser Ser Lys Val Gly Val PheThr Leu Cys Ile 65 70 75 80 Ala Ser Phe Leu Pro Ser Phe Val Leu His ValIle Ser Pro Ile Asn 85 90 95 Trp Asp Val Leu Arg Arg Lys Pro Gly Cys CysLeu Thr Lys Lys Asn 100 105 110 Glu Asn Thr Phe Asp Arg Arg Ile Ala GlyVal Thr Phe Tyr Arg Ser 115 120 125 Gln Met Met Leu Val Thr Val Thr CysIle Leu Ala Val Asp Phe Thr 130 135 140 Leu Phe Pro Arg Arg Tyr Ala LysVal Glu Thr Trp Gly Thr Ser Leu 145 150 155 160 Met Asp Leu Gly Val GlySer Phe Met Phe Ser Ser Gly Thr Val Ala 165 170 175 Gly Arg Lys Asn AspIle Lys Lys Pro Asn Ala Phe Lys Asn Val Leu 180 185 190 Trp Asn Ser PheIle Leu Leu Ile Leu Gly Phe Ala Arg Met Phe Leu 195 200 205 Thr Lys SerIle Asn Tyr Gln Glu His Val Ser Glu Tyr Gly Met His 210 215 220 Trp AsnPhe Phe Phe Thr Leu Gly Phe Met Ala Leu Gly Val Phe Phe 225 230 235 240Phe Arg Arg Ser Leu Lys Lys Val Ser Tyr Phe Asn Leu Ala Thr Phe 245 250255 Ile Thr Leu Leu His His Cys Leu Leu Val Leu Thr Pro Phe Gln Lys 260265 270 Trp Ala Leu Ser Ala Pro Arg Thr Asn Ile Leu Ala Gln Asn Arg Glu275 280 285 Gly Ile Ala Ser Leu Pro Gly Tyr Ile Ala Ile Tyr Phe Tyr GlyMet 290 295 300 Tyr Thr Gly Ser Val Val Leu Ala Asp Arg Pro Leu Met TyrThr Arg 305 310 315 320 Ala Glu Ser Trp Lys Arg Phe Gln Arg Leu Leu PhePro Leu Cys Ile 325 330 335 Leu Leu Val Leu Tyr Leu Val Ser Asn Phe LeuSer Val Gly Val Ser 340 345 350 Arg Arg Leu Ala Asn Thr Pro Tyr Val AlaAsn Val Ala Phe Ile Asn 355 360 365 Met Phe Phe Leu Thr Ile Tyr Ile LeuIle Asp Ala Tyr Leu Phe Pro 370 375 380 Ser Ser Val Pro Tyr Gly Ser ArgVal Pro Lys Leu Leu Glu Asp Ala 385 390 395 400 Asn Asn Asn Gly Leu LeuVal Phe Leu Ile Ala Asn Val Leu Thr Gly 405 410 415 Val Val Asn Leu SerPhe Asp Thr Leu His Ser Ser Asn Ala Lys Gly 420 425 430 Leu Thr Ile MetThr Met Tyr Leu Phe Ile Ile Cys Tyr Met Ala His 435 440 445 Trp Leu AlaGln His Gly Ile Arg Phe Arg Leu 450 455 29 35 DNA Artificial SequenceDescription of Artificial Sequenceprimer based on presumed DNA encodingGWT1 highly conserved region 29 gcnaargtng aracntgggg nacnwsnytn atgga35 30 38 DNA Artificial Sequence Description of ArtificialSequenceprimer based on presumed DNA encoding GWT1 highly conservedregion 30 ttccartgna ynccrtaytc ngtnacrtgy tcytgrta 38 31 32 DNAArtificial Sequence Description of Artificial Sequenceprimer based onpresumed DNA encoding GWT1 highly conserved region 31 gtraaraaraarttccartg naynccrtay tc 32 32 188 DNA Aspergillus fumigatus fragmenthaving homology to S. cerevisiae GWT1 32 atggatctgg gcgttggatcgtttgtcttt tcgggcggag tagtatccgc tcgctcacta 60 ctcaagagca ggaccaatggctctaaaagg ttgcctcttg ccaagaggtt gattgcgtcg 120 acgcgacact ctattcctctgctcgtcctc ggcctgattc ggctatacag cgtcaaaggc 180 ttggacta 188 33 24 DNAArtificial Sequence Description of Artificial SequencePCR amplificationprimer based on amplified fragment 33 ggagtagtat ccgctcgctc acta 24 3425 DNA Artificial Sequence Description of Artificial SequencePCRamplification primer based on amplified fragment 34 gtccaagcctttgacgctgt atagc 25 35 25 DNA Artificial Sequence Description ofArtificial SequencePCR amplification primer outside gene insertion siteof library 35 gggatgtgct gcaaggcgat taagt 25 36 26 DNA ArtificialSequence Description of Artificial SequencePCR amplification primeroutside gene insertion site of library 36 tttatgcttc cggctcgtat gttgtg26 37 25 DNA Artificial Sequence Description of ArtificialSequenceprimer corresponding to sequence upstream of initiation codon 37aaaggtgcaa atcccgcggc attga 25 38 28 DNA Artificial Sequence Descriptionof Artificial Sequenceprimer corresponding to sequence downstream ofstop codon 38 agttcactat atatcttcaa cacaccac 28 39 1576 DNA Aspergillusfumigatus amplified fragment of homologue of S. cerevisiae GWT1 fromcDNA library 39 aaggtgcaaa tcccgcggca ttgagtcaag atg gat cca gat tat aaagct cgc 54 Met Asp Pro Asp Tyr Lys Ala Arg 1 5 aaa gag gcc ttt gtc tcaggt ctt gca gga gga agc atc ctg gaa atc 102 Lys Glu Ala Phe Val Ser GlyLeu Ala Gly Gly Ser Ile Leu Glu Ile 10 15 20 aac gcc gtc acc ttg gtt gcttcg gta tcc gtt ttt ctg tgg tca att 150 Asn Ala Val Thr Leu Val Ala SerVal Ser Val Phe Leu Trp Ser Ile 25 30 35 40 cta caa tct cgc cta tcc tttttc aca ccc tac agc gcc gct gcc ctt 198 Leu Gln Ser Arg Leu Ser Phe PheThr Pro Tyr Ser Ala Ala Ala Leu 45 50 55 ctc gtt gat ttc ctg ctc aat gtacta gct atc ttg ttc gca acc act 246 Leu Val Asp Phe Leu Leu Asn Val LeuAla Ile Leu Phe Ala Thr Thr 60 65 70 tta tac tct tcg gcg cct ctt ctt ctcaat ctc ctt cta ata tct ccc 294 Leu Tyr Ser Ser Ala Pro Leu Leu Leu AsnLeu Leu Leu Ile Ser Pro 75 80 85 gct ctg ctg ata ctc ctc tct acg aaa cgtcct cgg acc ccc gtc aaa 342 Ala Leu Leu Ile Leu Leu Ser Thr Lys Arg ProArg Thr Pro Val Lys 90 95 100 gcg aaa cct cct cgc cag tcc gct aga gctggg aaa gat gac tcg aaa 390 Ala Lys Pro Pro Arg Gln Ser Ala Arg Ala GlyLys Asp Asp Ser Lys 105 110 115 120 cat gcg aca gcc ttg cca gag tct ctaccc att cat cca ttt ctc acg 438 His Ala Thr Ala Leu Pro Glu Ser Leu ProIle His Pro Phe Leu Thr 125 130 135 aca tat cgc gcc gcc atg atg gtt atcacg tgc atc gct atc ttg gct 486 Thr Tyr Arg Ala Ala Met Met Val Ile ThrCys Ile Ala Ile Leu Ala 140 145 150 gtg gat ttt cgc att ttt cct cgc cgattc gcc aag gta gaa aac tgg 534 Val Asp Phe Arg Ile Phe Pro Arg Arg PheAla Lys Val Glu Asn Trp 155 160 165 ggt aca tca ctc atg gat ctg ggc gttgga tcg ttt gtc ttt tcg ggc 582 Gly Thr Ser Leu Met Asp Leu Gly Val GlySer Phe Val Phe Ser Gly 170 175 180 gga gta gta tcc gct cgc tca cta ctcaag agc agg acc aat ggc tct 630 Gly Val Val Ser Ala Arg Ser Leu Leu LysSer Arg Thr Asn Gly Ser 185 190 195 200 aaa agg ttg cct ctt gcc aag aggttg att gcg tcg acg cga cac tct 678 Lys Arg Leu Pro Leu Ala Lys Arg LeuIle Ala Ser Thr Arg His Ser 205 210 215 att cct ctg ctc gtc ctc ggc ctgatt cgg cta tac agc gtc aaa ggc 726 Ile Pro Leu Leu Val Leu Gly Leu IleArg Leu Tyr Ser Val Lys Gly 220 225 230 ttg gac tat gcg gag cac gtc accgag tac ggc gta cat tgg aac ttc 774 Leu Asp Tyr Ala Glu His Val Thr GluTyr Gly Val His Trp Asn Phe 235 240 245 ttc ttt aca ttg ggt ctt ttg cctccg ttc gtg gag gtc ttc gac gcc 822 Phe Phe Thr Leu Gly Leu Leu Pro ProPhe Val Glu Val Phe Asp Ala 250 255 260 ttg gct acg atc att ccg tca tacgag gtt ctc tcc gtg ggg atc gcc 870 Leu Ala Thr Ile Ile Pro Ser Tyr GluVal Leu Ser Val Gly Ile Ala 265 270 275 280 gtc ttg tat caa gtt gcc ctagag tca aca gac ttg aaa agc tac atc 918 Val Leu Tyr Gln Val Ala Leu GluSer Thr Asp Leu Lys Ser Tyr Ile 285 290 295 ctc gtc tcc cct cgt ggg ccaagc tta ctg tcc aag aat cgt gaa ggc 966 Leu Val Ser Pro Arg Gly Pro SerLeu Leu Ser Lys Asn Arg Glu Gly 300 305 310 gtc ttc tcc ttc tca ggt tatctc gcg att ttt ctt gct ggt cgt gcg 1014 Val Phe Ser Phe Ser Gly Tyr LeuAla Ile Phe Leu Ala Gly Arg Ala 315 320 325 atc ggc att cgg ata atc cctcgc gga act tct ttc tca aga agc cca 1062 Ile Gly Ile Arg Ile Ile Pro ArgGly Thr Ser Phe Ser Arg Ser Pro 330 335 340 gaa cag gcc agg aga cgg gtcctg atc agc ctt ggc gtg caa gcg tta 1110 Glu Gln Ala Arg Arg Arg Val LeuIle Ser Leu Gly Val Gln Ala Leu 345 350 355 360 gtg tgg acc act ctt tttgtg ttg aac tcc act tat gcg atg gga tac 1158 Val Trp Thr Thr Leu Phe ValLeu Asn Ser Thr Tyr Ala Met Gly Tyr 365 370 375 gga gct aat atc cct gtctcc cgc cgc ctc gct aac atg ccc tat gtc 1206 Gly Ala Asn Ile Pro Val SerArg Arg Leu Ala Asn Met Pro Tyr Val 380 385 390 ctt tgg gtt tcg gcg ttcaac acc gcg caa ctg ttt gtg ttc tgc ctg 1254 Leu Trp Val Ser Ala Phe AsnThr Ala Gln Leu Phe Val Phe Cys Leu 395 400 405 atc gaa aca ctc tgc tttcct gca gtt cat cgg aca acg act caa gag 1302 Ile Glu Thr Leu Cys Phe ProAla Val His Arg Thr Thr Thr Gln Glu 410 415 420 agc gaa tct gag cga gtcgat ttt gct acg agc cga atc atg tcg gcc 1350 Ser Glu Ser Glu Arg Val AspPhe Ala Thr Ser Arg Ile Met Ser Ala 425 430 435 440 ttc aat aag aac agtctc gcg atc ttt ctt ttg gcc aat ctt ctg act 1398 Phe Asn Lys Asn Ser LeuAla Ile Phe Leu Leu Ala Asn Leu Leu Thr 445 450 455 gga gct gtg aat ctgagc atc tcc aca att gat gct aat aca gcg cag 1446 Gly Ala Val Asn Leu SerIle Ser Thr Ile Asp Ala Asn Thr Ala Gln 460 465 470 gcc atc gct gtt ctcatt gga tat tca tcc att atc aca ggg gtt gct 1494 Ala Ile Ala Val Leu IleGly Tyr Ser Ser Ile Ile Thr Gly Val Ala 475 480 485 cta gca ttg cat catgcc aat atc aaa gta ctt cct ttc tag 1536 Leu Ala Leu His His Ala Asn IleLys Val Leu Pro Phe 490 495 500 ggtatttacg agcaattggt ggtgtgttgaagatatatag 1576 40 501 PRT Aspergillus fumigatus homologue of S.cerevisiae GWT1 40 Met Asp Pro Asp Tyr Lys Ala Arg Lys Glu Ala Phe ValSer Gly Leu 1 5 10 15 Ala Gly Gly Ser Ile Leu Glu Ile Asn Ala Val ThrLeu Val Ala Ser 20 25 30 Val Ser Val Phe Leu Trp Ser Ile Leu Gln Ser ArgLeu Ser Phe Phe 35 40 45 Thr Pro Tyr Ser Ala Ala Ala Leu Leu Val Asp PheLeu Leu Asn Val 50 55 60 Leu Ala Ile Leu Phe Ala Thr Thr Leu Tyr Ser SerAla Pro Leu Leu 65 70 75 80 Leu Asn Leu Leu Leu Ile Ser Pro Ala Leu LeuIle Leu Leu Ser Thr 85 90 95 Lys Arg Pro Arg Thr Pro Val Lys Ala Lys ProPro Arg Gln Ser Ala 100 105 110 Arg Ala Gly Lys Asp Asp Ser Lys His AlaThr Ala Leu Pro Glu Ser 115 120 125 Leu Pro Ile His Pro Phe Leu Thr ThrTyr Arg Ala Ala Met Met Val 130 135 140 Ile Thr Cys Ile Ala Ile Leu AlaVal Asp Phe Arg Ile Phe Pro Arg 145 150 155 160 Arg Phe Ala Lys Val GluAsn Trp Gly Thr Ser Leu Met Asp Leu Gly 165 170 175 Val Gly Ser Phe ValPhe Ser Gly Gly Val Val Ser Ala Arg Ser Leu 180 185 190 Leu Lys Ser ArgThr Asn Gly Ser Lys Arg Leu Pro Leu Ala Lys Arg 195 200 205 Leu Ile AlaSer Thr Arg His Ser Ile Pro Leu Leu Val Leu Gly Leu 210 215 220 Ile ArgLeu Tyr Ser Val Lys Gly Leu Asp Tyr Ala Glu His Val Thr 225 230 235 240Glu Tyr Gly Val His Trp Asn Phe Phe Phe Thr Leu Gly Leu Leu Pro 245 250255 Pro Phe Val Glu Val Phe Asp Ala Leu Ala Thr Ile Ile Pro Ser Tyr 260265 270 Glu Val Leu Ser Val Gly Ile Ala Val Leu Tyr Gln Val Ala Leu Glu275 280 285 Ser Thr Asp Leu Lys Ser Tyr Ile Leu Val Ser Pro Arg Gly ProSer 290 295 300 Leu Leu Ser Lys Asn Arg Glu Gly Val Phe Ser Phe Ser GlyTyr Leu 305 310 315 320 Ala Ile Phe Leu Ala Gly Arg Ala Ile Gly Ile ArgIle Ile Pro Arg 325 330 335 Gly Thr Ser Phe Ser Arg Ser Pro Glu Gln AlaArg Arg Arg Val Leu 340 345 350 Ile Ser Leu Gly Val Gln Ala Leu Val TrpThr Thr Leu Phe Val Leu 355 360 365 Asn Ser Thr Tyr Ala Met Gly Tyr GlyAla Asn Ile Pro Val Ser Arg 370 375 380 Arg Leu Ala Asn Met Pro Tyr ValLeu Trp Val Ser Ala Phe Asn Thr 385 390 395 400 Ala Gln Leu Phe Val PheCys Leu Ile Glu Thr Leu Cys Phe Pro Ala 405 410 415 Val His Arg Thr ThrThr Gln Glu Ser Glu Ser Glu Arg Val Asp Phe 420 425 430 Ala Thr Ser ArgIle Met Ser Ala Phe Asn Lys Asn Ser Leu Ala Ile 435 440 445 Phe Leu LeuAla Asn Leu Leu Thr Gly Ala Val Asn Leu Ser Ile Ser 450 455 460 Thr IleAsp Ala Asn Thr Ala Gln Ala Ile Ala Val Leu Ile Gly Tyr 465 470 475 480Ser Ser Ile Ile Thr Gly Val Ala Leu Ala Leu His His Ala Asn Ile 485 490495 Lys Val Leu Pro Phe 500 41 1648 DNA Aspergillus fumigatus clonedhomologue of S. cerevisiae GWT1 from genomic library 41 gcaaatcccgcggcattgag tcaag atg gat cca gat tat aaa gct cgc aaa 52 Met Asp Pro AspTyr Lys Ala Arg Lys 1 5 gag gcc ttt gtc tca ggt ctt gca gga gga agc atcctg gaa atc aac 100 Glu Ala Phe Val Ser Gly Leu Ala Gly Gly Ser Ile LeuGlu Ile Asn 10 15 20 25 gcc gtc acc ttg gtt gct tcg gttcgtgttactatcttatt gtggctactt 151 Ala Val Thr Leu Val Ala Ser 30 cgcctacattgtttctcgac taaccgagtc tctttgcgat caatcag gta tcc gtt 207 Val Ser Val 35ttt ctg tgg tca att cta caa tct cgc cta tcc ttt ttc aca ccc tac 255 PheLeu Trp Ser Ile Leu Gln Ser Arg Leu Ser Phe Phe Thr Pro Tyr 40 45 50 agcgcc gct gcc ctt ctc gtt gat ttc ctg ctc aat gta cta gct atc 303 Ser AlaAla Ala Leu Leu Val Asp Phe Leu Leu Asn Val Leu Ala Ile 55 60 65 ttg ttcgca acc act tta tac tct tcg gcg cct ctt ctt ctc aat ctc 351 Leu Phe AlaThr Thr Leu Tyr Ser Ser Ala Pro Leu Leu Leu Asn Leu 70 75 80 ctt cta atatct ccc gct ctg ctg ata ctc ctc tct acg aaa cgt cct 399 Leu Leu Ile SerPro Ala Leu Leu Ile Leu Leu Ser Thr Lys Arg Pro 85 90 95 cgg acc ccc gtcaaa gcg aaa cct cct cgc cag tcc gct aga gct ggg 447 Arg Thr Pro Val LysAla Lys Pro Pro Arg Gln Ser Ala Arg Ala Gly 100 105 110 115 aaa gat gactcg aaa cat gcg aca gcc ttg cca gag tct cta ccc att 495 Lys Asp Asp SerLys His Ala Thr Ala Leu Pro Glu Ser Leu Pro Ile 120 125 130 cat cca tttctc acg aca tat cgc gcc gcc atg atg gtt atc acg tgc 543 His Pro Phe LeuThr Thr Tyr Arg Ala Ala Met Met Val Ile Thr Cys 135 140 145 atc gct atcttg gct gtg gat ttt cgc att ttt cct cgc cga ttc gcc 591 Ile Ala Ile LeuAla Val Asp Phe Arg Ile Phe Pro Arg Arg Phe Ala 150 155 160 aag gta gaaaac tgg ggt aca tca ctc atg gat ctg ggc gtt gga tcg 639 Lys Val Glu AsnTrp Gly Thr Ser Leu Met Asp Leu Gly Val Gly Ser 165 170 175 ttt gtc ttttcg ggc gga gta gta tcc gct cgc tca cta ctc aag agc 687 Phe Val Phe SerGly Gly Val Val Ser Ala Arg Ser Leu Leu Lys Ser 180 185 190 195 agg accaat ggc tct aaa agg ttg cct ctt gcc aag agg ttg att gcg 735 Arg Thr AsnGly Ser Lys Arg Leu Pro Leu Ala Lys Arg Leu Ile Ala 200 205 210 tcg acgcga cac tct att cct ctg ctc gtc ctc ggc ctg att cgg cta 783 Ser Thr ArgHis Ser Ile Pro Leu Leu Val Leu Gly Leu Ile Arg Leu 215 220 225 tac agcgtc aaa ggc ttg gac tat gcg gag cac gtc acc gag tac ggc 831 Tyr Ser ValLys Gly Leu Asp Tyr Ala Glu His Val Thr Glu Tyr Gly 230 235 240 gta cattgg aac ttc ttc ttt aca ttg ggt ctt ttg cct ccg ttc gtg 879 Val His TrpAsn Phe Phe Phe Thr Leu Gly Leu Leu Pro Pro Phe Val 245 250 255 gag gtcttc gac gcc ttg gct acg atc att ccg tca tac gag gtt ctc 927 Glu Val PheAsp Ala Leu Ala Thr Ile Ile Pro Ser Tyr Glu Val Leu 260 265 270 275 tccgtg ggg atc gcc gtc ttg tat caa gtt gcc cta gag tca aca gac 975 Ser ValGly Ile Ala Val Leu Tyr Gln Val Ala Leu Glu Ser Thr Asp 280 285 290 ttgaaa agc tac atc ctc gtc tcc cct cgt ggg cca agc tta ctg tcc 1023 Leu LysSer Tyr Ile Leu Val Ser Pro Arg Gly Pro Ser Leu Leu Ser 295 300 305 aagaat cgt gaa ggc gtc ttc tcc ttc tca ggt tat ctc gcg att ttt 1071 Lys AsnArg Glu Gly Val Phe Ser Phe Ser Gly Tyr Leu Ala Ile Phe 310 315 320 cttgct ggt cgt gcg atc ggc att cgg ata atc cct cgc gga act tct 1119 Leu AlaGly Arg Ala Ile Gly Ile Arg Ile Ile Pro Arg Gly Thr Ser 325 330 335 ttctca aga agc cca gaa cag gcc agg aga cgg gtc ctg atc agc ctt 1167 Phe SerArg Ser Pro Glu Gln Ala Arg Arg Arg Val Leu Ile Ser Leu 340 345 350 355ggc gtg caa gcg tta gtg tgg acc act ctt ttt gtg ttg aac tcc act 1215 GlyVal Gln Ala Leu Val Trp Thr Thr Leu Phe Val Leu Asn Ser Thr 360 365 370tat gcg atg gga tac gga gct aat atc cct gtc tcc cgc cgc ctc gct 1263 TyrAla Met Gly Tyr Gly Ala Asn Ile Pro Val Ser Arg Arg Leu Ala 375 380 385aac atg ccc tat gtc ctt tgg gtt tcg gcg ttc aac acc gcg caa ctg 1311 AsnMet Pro Tyr Val Leu Trp Val Ser Ala Phe Asn Thr Ala Gln Leu 390 395 400ttt gtg ttc tgc ctg atc gaa aca ctc tgc ttt cct gca gtt cat cgg 1359 PheVal Phe Cys Leu Ile Glu Thr Leu Cys Phe Pro Ala Val His Arg 405 410 415aca acg act caa gag agc gaa tct gag cga gtc gat ttt gct acg agc 1407 ThrThr Thr Gln Glu Ser Glu Ser Glu Arg Val Asp Phe Ala Thr Ser 420 425 430435 cga atc atg tcg gcc ttc aat aag aac agt ctc gcg atc ttt ctt ttg 1455Arg Ile Met Ser Ala Phe Asn Lys Asn Ser Leu Ala Ile Phe Leu Leu 440 445450 gcc aat ctt ctg act gga gct gtg aat ctg agc atc tcc aca att gat 1503Ala Asn Leu Leu Thr Gly Ala Val Asn Leu Ser Ile Ser Thr Ile Asp 455 460465 gct aat aca gcg cag gcc atc gct gtt ctc att gga tat tca tcc att 1551Ala Asn Thr Ala Gln Ala Ile Ala Val Leu Ile Gly Tyr Ser Ser Ile 470 475480 atc aca ggg gtt gct cta gca ttg cat cat gcc aat atc aaa gta ctt 1599Ile Thr Gly Val Ala Leu Ala Leu His His Ala Asn Ile Lys Val Leu 485 490495 cct ttc tag ggtatttacg agcaattggt ggtgtgttga agatatatag 1648 Pro Phe500 42 27 DNA Artificial Sequence Description of Artificial SequencePCRamplification primer 42 gccataataa gctaccgaat tgcaatg 27 43 26 DNAArtificial Sequence Description of Artificial SequencePCR amplificationprimer 43 cattaacacc cccattgaca accacg 26 44 1869 DNA Cryptococcusneoformans amplified fragment showing homology to S. cerevisiae GWT1 44ggggattaca agtcggccaa agaggccttt gtctcggata acccaggtgc ttctatctgg 60agtatcaacg ctgtcagcct ggtcgcactg gtatgtagct cgttctccga ggggttctgt 120catttggaga cgcttattaa ttgggatcgc aggcgacata tgctctctgg atcgccttat 180cgccgtacat ccgtcatgga ctcctgaaca actacctgat ctgtgttctt cccctattat 240tcggggtgac catcttctca acttcgcctc tcgtatttac ctcttttttg tccattattt 300ccctcgcttt catcacgaaa tcccaaaaat gcttcaaatc tgtcagttcg cccgaaaagc 360caaaaggcca atggctagac gaatcagact ccgatgagga accagcggaa cctgcttctg 420cagctggatc tgcagcagtc tcaccagtaa agcttctacc ttcccaagtg gcgttcgctt 480cgggatccct attatctccc gatccgacaa catcccccat gtcgccaagt agttcttcag 540cttcaggaca tgaagaccct ttggggatta tgggcgttaa cagacggagg tcgctattag 600aaggagtttc gcttgatgtt ccgtcacata tcgactccaa ggtcagaata tctcctgttc 660cctacttgag gctcaaaaag tctagggcaa cgaaggcgca atgggtgaaa gaaaagggaa 720gattaccatt tttgacagtg taccgagcgc acatgatgct catgactgtt atctgcatct 780tggcggtaga ttttgaagtg tttcctagat ggcagggcaa gtgcgaagat tttggtacta 840gtctggtaag ctttccttca gccatggtcc agtgctcacc gctctacttg ccgtagatgg 900acgtgggtgt cgggtcattc gtcttttccc tcggtctcgt ctccacaaaa tctctttctc 960ctccacctcc aactcctacg ccctcctcgc ccgctctcaa ctctcacatc attcccctca 1020ccccgtcccc gttcacttcc atcctcatct cgctccgaaa atccatcccc atcctcgtcc 1080tcggctttat acggttgatt atggtcaagg gatctgatta tcctgagcat gtgacggagt 1140acggcgtgca ctggaatttc ttcttcaccc tcgcattggt tcctgtgctc gccgtgggca 1200ttcgaccatt gacgcagtgg cttcgctgga gtgtgcttgg ggtaatcatc tctttgctgc 1260atcagctgtg gttaacatat tatctccaat ccatcgtctt ctcattcggc cggtcaggta 1320tctttctagc aaacaaggaa ggcttctcct ctcttcctgg ttatctttcc atatttttga 1380tcggcttgtc tattggagat catgttttaa ggctcagttt accaccaaga agagagaggg 1440tcgtgtcaga aacaaatgaa gagcatgagc agagtcattt tgagagaaaa aaattggatt 1500tgattatgga gttgattgga tatagcttag gctggtgggc actcttagga ggctggattt 1560gggccggcgg ggaggtatcc aggcgtttag taagtggaca tctttggtaa tattgtacct 1620atactaatcc ctgcataaag gccaacgctc cttatgtatt ttgggtagcg gcatacaata 1680ccacctttct cctcggctac ctcctcctta cccacattat tccatctccc acctcttccc 1740aaacatcacc atcgatctta gtgcctccct tgctcgacgc tatgaataaa aacggtctcg 1800cgatattttt ggcggccaac ttgcttacag gactggtgaa tgtgagcatg aagacaatgt 1860atgcgccgg 1869 45 27 DNA Artificial Sequence Description of ArtificialSequencePCR amplification primer 45 gtaaaggaag gcgctagaaa agatatg 27 4626 DNA Artificial Sequence Description of Artificial SequencePCRamplification primer 46 ctcatcggag tctgattcgt ctagcc 26 47 470 DNACryptococcus neoformans amplified fragment ovelapping SEQ ID NO44 47gaaggcgcta gaaaagatat ggtcttgtca tagcattaaa tccccgccat aataagctac 60tgaattgcaa tgggggatta caagtcggcc aaagaggcct ttgtctcgga taacccaggt 120gcttctatct ggagtatcaa cgctgtcagc ctggtcgcac tggtatgtag ctcgttctcc 180gaggggttct gtcatttgga gacgcttatt aattgggatc gcaggcgaca tatgctctct 240ggatcgcctt atcgccgtac atccgtcatg gactcctgaa caactacctg atctgtgttc 300ttcccctatt attcggggtg accatcttct caacttcgcc tctcgtattt acctcttttt 360tgtccattat ttccctcgct ttcatcacga aatcccaaaa atgcttcaaa tctgtcagtt 420cgcccgaaaa gccaaaaggc caatggctag acgaatcaga ctccgatgag 470 48 37 DNAArtificial Sequence Description of Artificial Sequence3′-RACEadaptor-primer 48 gcccacgcgt cgactagtac tttttttttt ttttttt 37 49 29 DNAArtificial Sequence Description of Artificial SequencePCR amplificationprimer 49 catcttggcg gtagattttg aagtgttcc 29 50 20 DNA ArtificialSequence Description of Artificial SequencePCR amplification primer 50ggccacgcgt cgactagtac 20 51 1136 DNA Cryptococcus neoformans amplifiedfragment showing homology to S. cerevisiae GWT1 51 gcggtagatt ttgaagtgttccctagatgg cagggcaagt gcgaagattt tggtactagt 60 ctgatggacg tgggtgtcgggtcattcgtc ttttccctcg gtctcgtctc cacaaaatct 120 ctttctcctc cacctccaactcctacgccc tcctcgcccg ctctcaactc tcacatcatt 180 cccctcaccc cgtccccgttcacttccatc ctcatctcgc tccgaaaatc catccccatc 240 ctcgtcctcg gctttatacggttgattatg gtcaagggat ctgattatcc tgagcatgtg 300 acggagtacg gcgtgcactggaatttcttc ttcaccctcg cattggttcc tgtgctcgcc 360 gtgggcattc gaccattgacgcagtggctt cgctggagtg tgcttggggt aatcatctct 420 ttgctgcatc agctgtggttaacatattat ctccaatcca tcgtcttctc attcggccgg 480 tcaggtatct ttctagcaaacaaggaaggc ttctcctctc ttcctggtta tctttccata 540 tttttgatcg gcttgtctattggagatcat gttttaaggc tcagtttacc accaagaaga 600 gagagggtcg tgtcagaaacaaatgaagag catgagcaga gtcattttga gagaaaaaaa 660 ttggatttga ttatggagttgattggatat agcttaggct ggtgggcact cttaggaggc 720 tggatttggg ccggcggggaggtatccagg cgtttagcca acgctcctta tgtattttgg 780 gtagcggcat acaataccacctttctcctc ggctacctcc tccttaccca cattattcca 840 tctcccacct cttcccaaacatcaccatcg atcttagtgc ctcccttgct cgacgctatg 900 aataaaaacg gtctcgcgatatttttggcg gccaacttgc ttacaggact ggtgaatgtg 960 agcatgaaga caatgtatgcgccggcgtgg ttgtcaatgg gggtgttaat gttgtatacc 1020 ttgacaatca gttgtgtagggtggatactg aaaggacgga ggatcaagat atagttaaag 1080 tgtttaccat gcaggatactgagtatctcg gttcaaaaaa aaaaaaaaaa aaaaaa 1136 52 27 DNA ArtificialSequence Description of Artificial SequencePCR amplification primer 52gtcttgtcat agcattaaat ccccgcc 27 53 28 DNA Artificial SequenceDescription of Artificial SequencePCR amplification primer 53 gaaccgagatactcagtatc ctgcatgg 28 54 2045 DNA Cryptococcus neoformans full lengthhomologue of S. cerevisiae GWT1 54 gtcatagcat taaatccccg ccataataagctactgaatt gca atg ggg gat tac 55 Met Gly Asp Tyr 1 aag tcg gcc aaa gaggcc ttt gtc tcg gat aac cca ggt gct tct atc 103 Lys Ser Ala Lys Glu AlaPhe Val Ser Asp Asn Pro Gly Ala Ser Ile 5 10 15 20 tgg agt atc aac gctgtc agc ctg gtc gca ctg gtatgtagct cgttctccga 156 Trp Ser Ile Asn AlaVal Ser Leu Val Ala Leu 25 30 ggggttctgt catttggaga cgcttattaattgggatcgc ag gcg aca tat gct 210 Ala Thr Tyr Ala 35 ctc tgg atc gcc ttatcg ccg tac atc cgt cat gga ctc ctg aac aac 258 Leu Trp Ile Ala Leu SerPro Tyr Ile Arg His Gly Leu Leu Asn Asn 40 45 50 tac ctg atc tgt gtt cttccc cta tta ttc ggg gtg acc atc ttc tca 306 Tyr Leu Ile Cys Val Leu ProLeu Leu Phe Gly Val Thr Ile Phe Ser 55 60 65 act tcg cct ctc gta ttt acctct ttt ttg tcc att att tcc ctc gct 354 Thr Ser Pro Leu Val Phe Thr SerPhe Leu Ser Ile Ile Ser Leu Ala 70 75 80 ttc atc acg aaa tcc caa aaa tgcttc aaa tct gtc agt tcg ccc gaa 402 Phe Ile Thr Lys Ser Gln Lys Cys PheLys Ser Val Ser Ser Pro Glu 85 90 95 aag cca aaa ggc caa tgg cta gac gaatca gac tcc gat gag gaa cca 450 Lys Pro Lys Gly Gln Trp Leu Asp Glu SerAsp Ser Asp Glu Glu Pro 100 105 110 115 gcg gaa cct gct tct gca gct ggatct gca gca gtc tca cca gta aag 498 Ala Glu Pro Ala Ser Ala Ala Gly SerAla Ala Val Ser Pro Val Lys 120 125 130 ctt cta cct tcc caa gtg gcg ttcgct tcg gga tcc cta tta tct ccc 546 Leu Leu Pro Ser Gln Val Ala Phe AlaSer Gly Ser Leu Leu Ser Pro 135 140 145 gat ccg aca aca tcc ccc atg tcgcca agt agt tct tca gct tca gga 594 Asp Pro Thr Thr Ser Pro Met Ser ProSer Ser Ser Ser Ala Ser Gly 150 155 160 cat gaa gac cct ttg ggg att atgggc gtt aac aga cgg agg tcg cta 642 His Glu Asp Pro Leu Gly Ile Met GlyVal Asn Arg Arg Arg Ser Leu 165 170 175 tta gaa gga gtt tcg ctt gat gttccg tca cat atc gac tcc aag gtc 690 Leu Glu Gly Val Ser Leu Asp Val ProSer His Ile Asp Ser Lys Val 180 185 190 195 aga ata tct cct gtt ccc tacttg agg ctc aaa aag tct agg gca acg 738 Arg Ile Ser Pro Val Pro Tyr LeuArg Leu Lys Lys Ser Arg Ala Thr 200 205 210 aag gcg caa tgg gtg aaa gaaaag gga aga tta cca ttt ttg aca gtg 786 Lys Ala Gln Trp Val Lys Glu LysGly Arg Leu Pro Phe Leu Thr Val 215 220 225 tac cga gcg cac atg atg ctcatg act gtt atc tgc atc ttg gcg gta 834 Tyr Arg Ala His Met Met Leu MetThr Val Ile Cys Ile Leu Ala Val 230 235 240 gat ttt gaa gtg ttt cct agatgg cag ggc aag tgc gaa gat ttt ggt 882 Asp Phe Glu Val Phe Pro Arg TrpGln Gly Lys Cys Glu Asp Phe Gly 245 250 255 act agt ctg gtaagctttccttcagccat ggtccagtgc tcaccgctct 931 Thr Ser Leu 260 acttgccgta g atggac gtg ggt gtc ggg tca ttc gtc ttt tcc ctc ggt 981 Met Asp Val Gly ValGly Ser Phe Val Phe Ser Leu Gly 265 270 275 ctc gtc tcc aca aaa tct ctttct cct cca cct cca act cct acg ccc 1029 Leu Val Ser Thr Lys Ser Leu SerPro Pro Pro Pro Thr Pro Thr Pro 280 285 290 tcc tcg ccc gct ctc aac tctcac atc att ccc ctc acc ccg tcc ccg 1077 Ser Ser Pro Ala Leu Asn Ser HisIle Ile Pro Leu Thr Pro Ser Pro 295 300 305 ttc act tcc atc ctc atc tcgctc cga aaa tcc atc ccc atc ctc gtc 1125 Phe Thr Ser Ile Leu Ile Ser LeuArg Lys Ser Ile Pro Ile Leu Val 310 315 320 ctc ggc ttt ata cgg ttg attatg gtc aag gga tct gat tat cct gag 1173 Leu Gly Phe Ile Arg Leu Ile MetVal Lys Gly Ser Asp Tyr Pro Glu 325 330 335 cat gtg acg gag tac ggc gtgcac tgg aat ttc ttc ttc acc ctc gca 1221 His Val Thr Glu Tyr Gly Val HisTrp Asn Phe Phe Phe Thr Leu Ala 340 345 350 355 ttg gtt cct gtg ctc gccgtg ggc att cga cca ttg acg cag tgg ctt 1269 Leu Val Pro Val Leu Ala ValGly Ile Arg Pro Leu Thr Gln Trp Leu 360 365 370 cgc tgg agt gtg ctt ggggta atc atc tct ttg ctg cat cag ctg tgg 1317 Arg Trp Ser Val Leu Gly ValIle Ile Ser Leu Leu His Gln Leu Trp 375 380 385 tta aca tat tat ctc caatcc atc gtc ttc tca ttc ggc cgg tca ggt 1365 Leu Thr Tyr Tyr Leu Gln SerIle Val Phe Ser Phe Gly Arg Ser Gly 390 395 400 atc ttt cta gca aac aaggaa ggc ttc tcc tct ctt cct ggt tat ctt 1413 Ile Phe Leu Ala Asn Lys GluGly Phe Ser Ser Leu Pro Gly Tyr Leu 405 410 415 tcc ata ttt ttg atc ggcttg tct att gga gat cat gtt tta agg ctc 1461 Ser Ile Phe Leu Ile Gly LeuSer Ile Gly Asp His Val Leu Arg Leu 420 425 430 435 agt tta cca cca agaaga gag agg gtc gtg tca gaa aca aat gaa gag 1509 Ser Leu Pro Pro Arg ArgGlu Arg Val Val Ser Glu Thr Asn Glu Glu 440 445 450 cat gag cag agt catttt gag aga aaa aaa ttg gat ttg att atg gag 1557 His Glu Gln Ser His PheGlu Arg Lys Lys Leu Asp Leu Ile Met Glu 455 460 465 ttg att gga tat agctta ggc tgg tgg gca ctc tta gga ggc tgg att 1605 Leu Ile Gly Tyr Ser LeuGly Trp Trp Ala Leu Leu Gly Gly Trp Ile 470 475 480 tgg gcc ggc ggg gaggta tcc agg cgt tta gtaagtggac atctttggta 1655 Trp Ala Gly Gly Glu ValSer Arg Arg Leu 485 490 atattgtacc tatactaatc cctgcataaa g gcc aac gctcct tat gta ttt 1707 Ala Asn Ala Pro Tyr Val Phe 495 500 tgg gta gcg gcatac aat acc acc ttt ctc ctc ggc tac ctc ctc ctt 1755 Trp Val Ala Ala TyrAsn Thr Thr Phe Leu Leu Gly Tyr Leu Leu Leu 505 510 515 acc cac att attcca tct ccc acc tct tcc caa aca tca cca tcg atc 1803 Thr His Ile Ile ProSer Pro Thr Ser Ser Gln Thr Ser Pro Ser Ile 520 525 530 tta gtg cct cccttg ctc gac gct atg aat aaa aac ggt ctc gcg ata 1851 Leu Val Pro Pro LeuLeu Asp Ala Met Asn Lys Asn Gly Leu Ala Ile 535 540 545 ttt ttg gcg gccaac ttg ctt aca gga ctg gtg aat gtg agc atg aag 1899 Phe Leu Ala Ala AsnLeu Leu Thr Gly Leu Val Asn Val Ser Met Lys 550 555 560 aca atg tat gcgccg gcg tgg ttg tca atg ggg gtg tta atg ttg tat 1947 Thr Met Tyr Ala ProAla Trp Leu Ser Met Gly Val Leu Met Leu Tyr 565 570 575 580 acc ttg acaatc agt tgt gta ggg tgg ata ctg aaa gga cgg agg atc 1995 Thr Leu Thr IleSer Cys Val Gly Trp Ile Leu Lys Gly Arg Arg Ile 585 590 595 aag ata tagttaaagtgtt taccatgcag gatactgagt atctcggttc a 2045 Lys Ile 55 23 DNAArtificial Sequence Description of Artificial SequencePCR amplificationprimer predicted to be junction between exons 55 cagcctggtc gcactggcgacat 23 56 25 DNA Artificial Sequence Description of ArtificialSequencePCR amplification primer predicted to be junction between exons56 cataaggagc gttggctaaa cgcct 25 57 1418 DNA Cryptococcus neoformansamplified fragment showing homology to S. cerevisiae GWT1 57 cagcctggtcgcactggcga catatgctct ctggatcgcc ttatcgccgt acatccgtca 60 tggactcctgaacaactacc tgatctgtgt tcttccccta ttattcgggg tgaccatctt 120 ctcaacttcgcctctcgtat ttacctcttt tttgtccatt atttccctcg ctttcatcac 180 gaaatcccaaaaatgcttca aatctgtcag ttcgcccgaa aagccaaaag gccaatggct 240 agacgaatcagactccgatg aggaaccagc ggaacctgct tctgcagctg gatctgcagc 300 agtctcaccagtaaagcttc taccttccca agtggcgttc gcttcgggat ccctattatc 360 tcccgatccgacaacatccc ccatgtcgcc aagtagttct tcagcttcag gacatgaaga 420 ccctttggggattatgggcg ttaacagacg gaggtcgcta ttagaaggag tttcgcttga 480 tgttccgtcacatatcgact ccaaggtcag aatatctcct gttccctact tgaggctcaa 540 aaagtctagggcaacgaagg cgcaatgggt gaaagaaaag ggaagattac catttttgac 600 agtgtaccgagcgcacatga tgctcatgac tgttatctgc atcttggcgg tagattttga 660 agtgtttcctagatggcagg gcaagtgcga agattttggt actagtctga tggacgtggg 720 tgtcgggtcattcgtctttt ccctcggtct cgtctccaca aaatctcttt ctcctccacc 780 tccaactcctacgccctcct cgcccgctct caactctcac atcattcccc tcaccccgtc 840 cccgttcacttccatcctca tctcgctccg aaaatccatc cccatcctcg tcctcggctt 900 tatacggttgattatggtca agggatctga ttatcctgag catgtgacgg agtacggcgt 960 gcactggaatttcttcttca ccctcgcatt ggttcctgtg ctcgccgtgg gcattcgacc 1020 attgacgcagtggcttcgct ggagtgtgct tggggtaatc atctctttgc tgcatcagct 1080 gtggttaacatattatctcc aatccatcgt cttctcattc ggccggtcag gtatctttct 1140 agcaaacaaggaaggcttct cctctcttcc tggttatctt tccatatttt tgatcggctt 1200 gtctattggagatcatgttt taaggctcag tttaccacca agaagagaga gggtcgtgtc 1260 agaaacaaatgaagagcatg agcagagtca ttttgagaga aaaaaattgg atttgattat 1320 ggagttgattggatatagct taggctggtg ggcactctta ggaggctgga tttgggccgg 1380 cggggaggtatccaggcgtt tagccaacgc tccttatg 1418 58 1797 DNA Cryptococcus neoformansCDS (1)..(1797) homologue of S. cerevisiae GWT1 58 atg ggg gat tac aagtcg gcc aaa gag gcc ttt gtc tcg gat aac cca 48 Met Gly Asp Tyr Lys SerAla Lys Glu Ala Phe Val Ser Asp Asn Pro 1 5 10 15 ggt gct tct atc tggagt atc aac gct gtc agc ctg gtc gca ctg gcg 96 Gly Ala Ser Ile Trp SerIle Asn Ala Val Ser Leu Val Ala Leu Ala 20 25 30 aca tat gct ctc tgg atcgcc tta tcg ccg tac atc cgt cat gga ctc 144 Thr Tyr Ala Leu Trp Ile AlaLeu Ser Pro Tyr Ile Arg His Gly Leu 35 40 45 ctg aac aac tac ctg atc tgtgtt ctt ccc cta tta ttc ggg gtg acc 192 Leu Asn Asn Tyr Leu Ile Cys ValLeu Pro Leu Leu Phe Gly Val Thr 50 55 60 atc ttc tca act tcg cct ctc gtattt acc tct ttt ttg tcc att att 240 Ile Phe Ser Thr Ser Pro Leu Val PheThr Ser Phe Leu Ser Ile Ile 65 70 75 80 tcc ctc gct ttc atc acg aaa tcccaa aaa tgc ttc aaa tct gtc agt 288 Ser Leu Ala Phe Ile Thr Lys Ser GlnLys Cys Phe Lys Ser Val Ser 85 90 95 tcg ccc gaa aag cca aaa ggc caa tggcta gac gaa tca gac tcc gat 336 Ser Pro Glu Lys Pro Lys Gly Gln Trp LeuAsp Glu Ser Asp Ser Asp 100 105 110 gag gaa cca gcg gaa cct gct tct gcagct gga tct gca gca gtc tca 384 Glu Glu Pro Ala Glu Pro Ala Ser Ala AlaGly Ser Ala Ala Val Ser 115 120 125 cca gta aag ctt cta cct tcc caa gtggcg ttc gct tcg gga tcc cta 432 Pro Val Lys Leu Leu Pro Ser Gln Val AlaPhe Ala Ser Gly Ser Leu 130 135 140 tta tct ccc gat ccg aca aca tcc cccatg tcg cca agt agt tct tca 480 Leu Ser Pro Asp Pro Thr Thr Ser Pro MetSer Pro Ser Ser Ser Ser 145 150 155 160 gct tca gga cat gaa gac cct ttgggg att atg ggc gtt aac aga cgg 528 Ala Ser Gly His Glu Asp Pro Leu GlyIle Met Gly Val Asn Arg Arg 165 170 175 agg tcg cta tta gaa gga gtt tcgctt gat gtt ccg tca cat atc gac 576 Arg Ser Leu Leu Glu Gly Val Ser LeuAsp Val Pro Ser His Ile Asp 180 185 190 tcc aag gtc aga ata tct cct gttccc tac ttg agg ctc aaa aag tct 624 Ser Lys Val Arg Ile Ser Pro Val ProTyr Leu Arg Leu Lys Lys Ser 195 200 205 agg gca acg aag gcg caa tgg gtgaaa gaa aag gga aga tta cca ttt 672 Arg Ala Thr Lys Ala Gln Trp Val LysGlu Lys Gly Arg Leu Pro Phe 210 215 220 ttg aca gtg tac cga gcg cac atgatg ctc atg act gtt atc tgc atc 720 Leu Thr Val Tyr Arg Ala His Met MetLeu Met Thr Val Ile Cys Ile 225 230 235 240 ttg gcg gta gat ttt gaa gtgttt cct aga tgg cag ggc aag tgc gaa 768 Leu Ala Val Asp Phe Glu Val PhePro Arg Trp Gln Gly Lys Cys Glu 245 250 255 gat ttt ggt act agt ctg atggac gtg ggt gtc ggg tca ttc gtc ttt 816 Asp Phe Gly Thr Ser Leu Met AspVal Gly Val Gly Ser Phe Val Phe 260 265 270 tcc ctc ggt ctc gtc tcc acaaaa tct ctt tct cct cca cct cca act 864 Ser Leu Gly Leu Val Ser Thr LysSer Leu Ser Pro Pro Pro Pro Thr 275 280 285 cct acg ccc tcc tcg ccc gctctc aac tct cac atc att ccc ctc acc 912 Pro Thr Pro Ser Ser Pro Ala LeuAsn Ser His Ile Ile Pro Leu Thr 290 295 300 ccg tcc ccg ttc act tcc atcctc atc tcg ctc cga aaa tcc atc ccc 960 Pro Ser Pro Phe Thr Ser Ile LeuIle Ser Leu Arg Lys Ser Ile Pro 305 310 315 320 atc ctc gtc ctc ggc tttata cgg ttg att atg gtc aag gga tct gat 1008 Ile Leu Val Leu Gly Phe IleArg Leu Ile Met Val Lys Gly Ser Asp 325 330 335 tat cct gag cat gtg acggag tac ggc gtg cac tgg aat ttc ttc ttc 1056 Tyr Pro Glu His Val Thr GluTyr Gly Val His Trp Asn Phe Phe Phe 340 345 350 acc ctc gca ttg gtt cctgtg ctc gcc gtg ggc att cga cca ttg acg 1104 Thr Leu Ala Leu Val Pro ValLeu Ala Val Gly Ile Arg Pro Leu Thr 355 360 365 cag tgg ctt cgc tgg agtgtg ctt ggg gta atc atc tct ttg ctg cat 1152 Gln Trp Leu Arg Trp Ser ValLeu Gly Val Ile Ile Ser Leu Leu His 370 375 380 cag ctg tgg tta aca tattat ctc caa tcc atc gtc ttc tca ttc ggc 1200 Gln Leu Trp Leu Thr Tyr TyrLeu Gln Ser Ile Val Phe Ser Phe Gly 385 390 395 400 cgg tca ggt atc tttcta gca aac aag gaa ggc ttc tcc tct ctt cct 1248 Arg Ser Gly Ile Phe LeuAla Asn Lys Glu Gly Phe Ser Ser Leu Pro 405 410 415 ggt tat ctt tcc atattt ttg atc ggc ttg tct att gga gat cat gtt 1296 Gly Tyr Leu Ser Ile PheLeu Ile Gly Leu Ser Ile Gly Asp His Val 420 425 430 tta agg ctc agt ttacca cca aga aga gag agg gtc gtg tca gaa aca 1344 Leu Arg Leu Ser Leu ProPro Arg Arg Glu Arg Val Val Ser Glu Thr 435 440 445 aat gaa gag cat gagcag agt cat ttt gag aga aaa aaa ttg gat ttg 1392 Asn Glu Glu His Glu GlnSer His Phe Glu Arg Lys Lys Leu Asp Leu 450 455 460 att atg gag ttg attgga tat agc tta ggc tgg tgg gca ctc tta gga 1440 Ile Met Glu Leu Ile GlyTyr Ser Leu Gly Trp Trp Ala Leu Leu Gly 465 470 475 480 ggc tgg att tgggcc ggc ggg gag gta tcc agg cgt tta gcc aac gct 1488 Gly Trp Ile Trp AlaGly Gly Glu Val Ser Arg Arg Leu Ala Asn Ala 485 490 495 cct tat gta ttttgg gta gcg gca tac aat acc acc ttt ctc ctc ggc 1536 Pro Tyr Val Phe TrpVal Ala Ala Tyr Asn Thr Thr Phe Leu Leu Gly 500 505 510 tac ctc ctc cttacc cac att att cca tct ccc acc tct tcc caa aca 1584 Tyr Leu Leu Leu ThrHis Ile Ile Pro Ser Pro Thr Ser Ser Gln Thr 515 520 525 tca cca tcg atctta gtg cct ccc ttg ctc gac gct atg aat aaa aac 1632 Ser Pro Ser Ile LeuVal Pro Pro Leu Leu Asp Ala Met Asn Lys Asn 530 535 540 ggt ctc gcg atattt ttg gcg gcc aac ttg ctt aca gga ctg gtg aat 1680 Gly Leu Ala Ile PheLeu Ala Ala Asn Leu Leu Thr Gly Leu Val Asn 545 550 555 560 gtg agc atgaag aca atg tat gcg ccg gcg tgg ttg tca atg ggg gtg 1728 Val Ser Met LysThr Met Tyr Ala Pro Ala Trp Leu Ser Met Gly Val 565 570 575 tta atg ttgtat acc ttg aca atc agt tgt gta ggg tgg ata ctg aaa 1776 Leu Met Leu TyrThr Leu Thr Ile Ser Cys Val Gly Trp Ile Leu Lys 580 585 590 gga cgg aggatc aag ata tag 1797 Gly Arg Arg Ile Lys Ile 595 59 598 PRT Cryptococcusneoformans homologue of S. cerevisiae GWT1 59 Met Gly Asp Tyr Lys SerAla Lys Glu Ala Phe Val Ser Asp Asn Pro 1 5 10 15 Gly Ala Ser Ile TrpSer Ile Asn Ala Val Ser Leu Val Ala Leu Ala 20 25 30 Thr Tyr Ala Leu TrpIle Ala Leu Ser Pro Tyr Ile Arg His Gly Leu 35 40 45 Leu Asn Asn Tyr LeuIle Cys Val Leu Pro Leu Leu Phe Gly Val Thr 50 55 60 Ile Phe Ser Thr SerPro Leu Val Phe Thr Ser Phe Leu Ser Ile Ile 65 70 75 80 Ser Leu Ala PheIle Thr Lys Ser Gln Lys Cys Phe Lys Ser Val Ser 85 90 95 Ser Pro Glu LysPro Lys Gly Gln Trp Leu Asp Glu Ser Asp Ser Asp 100 105 110 Glu Glu ProAla Glu Pro Ala Ser Ala Ala Gly Ser Ala Ala Val Ser 115 120 125 Pro ValLys Leu Leu Pro Ser Gln Val Ala Phe Ala Ser Gly Ser Leu 130 135 140 LeuSer Pro Asp Pro Thr Thr Ser Pro Met Ser Pro Ser Ser Ser Ser 145 150 155160 Ala Ser Gly His Glu Asp Pro Leu Gly Ile Met Gly Val Asn Arg Arg 165170 175 Arg Ser Leu Leu Glu Gly Val Ser Leu Asp Val Pro Ser His Ile Asp180 185 190 Ser Lys Val Arg Ile Ser Pro Val Pro Tyr Leu Arg Leu Lys LysSer 195 200 205 Arg Ala Thr Lys Ala Gln Trp Val Lys Glu Lys Gly Arg LeuPro Phe 210 215 220 Leu Thr Val Tyr Arg Ala His Met Met Leu Met Thr ValIle Cys Ile 225 230 235 240 Leu Ala Val Asp Phe Glu Val Phe Pro Arg TrpGln Gly Lys Cys Glu 245 250 255 Asp Phe Gly Thr Ser Leu Met Asp Val GlyVal Gly Ser Phe Val Phe 260 265 270 Ser Leu Gly Leu Val Ser Thr Lys SerLeu Ser Pro Pro Pro Pro Thr 275 280 285 Pro Thr Pro Ser Ser Pro Ala LeuAsn Ser His Ile Ile Pro Leu Thr 290 295 300 Pro Ser Pro Phe Thr Ser IleLeu Ile Ser Leu Arg Lys Ser Ile Pro 305 310 315 320 Ile Leu Val Leu GlyPhe Ile Arg Leu Ile Met Val Lys Gly Ser Asp 325 330 335 Tyr Pro Glu HisVal Thr Glu Tyr Gly Val His Trp Asn Phe Phe Phe 340 345 350 Thr Leu AlaLeu Val Pro Val Leu Ala Val Gly Ile Arg Pro Leu Thr 355 360 365 Gln TrpLeu Arg Trp Ser Val Leu Gly Val Ile Ile Ser Leu Leu His 370 375 380 GlnLeu Trp Leu Thr Tyr Tyr Leu Gln Ser Ile Val Phe Ser Phe Gly 385 390 395400 Arg Ser Gly Ile Phe Leu Ala Asn Lys Glu Gly Phe Ser Ser Leu Pro 405410 415 Gly Tyr Leu Ser Ile Phe Leu Ile Gly Leu Ser Ile Gly Asp His Val420 425 430 Leu Arg Leu Ser Leu Pro Pro Arg Arg Glu Arg Val Val Ser GluThr 435 440 445 Asn Glu Glu His Glu Gln Ser His Phe Glu Arg Lys Lys LeuAsp Leu 450 455 460 Ile Met Glu Leu Ile Gly Tyr Ser Leu Gly Trp Trp AlaLeu Leu Gly 465 470 475 480 Gly Trp Ile Trp Ala Gly Gly Glu Val Ser ArgArg Leu Ala Asn Ala 485 490 495 Pro Tyr Val Phe Trp Val Ala Ala Tyr AsnThr Thr Phe Leu Leu Gly 500 505 510 Tyr Leu Leu Leu Thr His Ile Ile ProSer Pro Thr Ser Ser Gln Thr 515 520 525 Ser Pro Ser Ile Leu Val Pro ProLeu Leu Asp Ala Met Asn Lys Asn 530 535 540 Gly Leu Ala Ile Phe Leu AlaAla Asn Leu Leu Thr Gly Leu Val Asn 545 550 555 560 Val Ser Met Lys ThrMet Tyr Ala Pro Ala Trp Leu Ser Met Gly Val 565 570 575 Leu Met Leu TyrThr Leu Thr Ile Ser Cys Val Gly Trp Ile Leu Lys 580 585 590 Gly Arg ArgIle Lys Ile 595 60 30 DNA Artificial Sequence Description of ArtificialSequenceR1 and R5 mutant GWT1 gene primer 60 aaagaattca tggcaacagtacatcagaag 30 61 20 DNA Artificial Sequence Description of ArtificialSequenceR1 and R5 mutant GWT1 gene primer 61 gggcactgtt gaaaaaccta 20 621428 DNA Saccharomyces cerevisiae promoter (1)..(1428) GWT1 promoterregion 62 gttgttcaaa atgggggtaa aattgagacg tcttacttga gcggcatttacgatcattct 60 tattacatca ttccaagtaa taaagctctt gactccttca atgatttacctgagattata 120 gatgataatg atggtatagt tacagaattt ttcattgaac gctgcttgtattatcaaaaa 180 ttactacacc caatagattt atggtcaaaa cccttcctca gcacaatagagtttcaagtt 240 tcgtcttctt caaagttatt gcatcatgaa ttttcttctt ccccttttctgaatgttact 300 atcactggat tctctggcgt agagctgtta catctgacta aagtattaaatcttctaaaa 360 ccaatgggca tcaattatgt agaatacctc aataaatcca ctgacattctgctaatcaac 420 ttagcagctt tacccagtat cccgaaaacc catccgttat ggtcgaatgaatttagcgat 480 ctttttactc agttttgcat taataacaat aatgatgatc ctggtgataataacagaaaa 540 gattttcaaa ataattcaat cttgagaaat tcgatgaaaa ggaaaattgaatatatcaag 600 aaattccact ccataccggt agttactcca gcatttattt ttaaattattgtccgctgca 660 tctggagaaa ataatgaaat ctttttaaac aatatcaagt ggtgtattatctgcccaaga 720 ggacacaagg acgattttaa atgtaagata aaaaaaccat actataccagcattagttca 780 gaaaaaaagt accaaaacaa tgatccaaaa atcgacaaaa ctattcttttgaaaagaaac 840 aattcctcat tatcggagca ctctatgaaa gataccaaaa acgaattattgcagaaaatt 900 agagaaactg attctggaag aaaaaagcgt agtgtctcat cgagtatcatggatgtttct 960 tcagagagac aaatgccgga tacgaaaagg atcaagttgg agtcactgccaaaaaatttc 1020 gttcctaaac aaattaaacg aaccacgagt tggggcacaa taatgtcagaaaatgtgcct 1080 acagagcagc cgactgcaat ttctaatcca gaagagatcc caagaactgaggaagtttca 1140 catactcaag ttacctatgg ctccattcaa gataagaaac gtactgcctctttagaaaaa 1200 cctatgagac gacagacaag aaatcagaca aaggaattag attcttgaaatgtagtccgt 1260 aattttataa gatattcatt tacatacgcc atctacagca ttattcaaatctactcatct 1320 atatgtatta ccgttttgta tgataatact ttccatgaca tgctcgcgtgaaaaaacagc 1380 atgagaaaaa gaggatcgca ataagaagac acgtaaatat ctaaataa1428 63 133 DNA Saccharomyces cerevisiae terminator (1)..(133) GWT1terminator region 63 taacacacca tccacatttc catgtagttc gtatacaaaccctaccagta aaataaaatt 60 aactcctatg tgctttaaat aaaaattata aaccgcctccaatagttgac gtagtcaggc 120 atgaaagtgc tac 133 64 31 PRT Saccharomycescerevisiae highly conserved GWT1 F-domain 64 Ile Leu Ala Val Asp Phe ProIle Phe Pro Arg Arg Phe Ala Lys Val 1 5 10 15 Glu Thr Trp Gly Thr SerLeu Met Asp Leu Gly Val Gly Ser Phe 20 25 30 65 31 PRT Candida albicanshighly conserved GWT1 F-domain 65 Ile Leu Ala Val Asp Phe Pro Ile PhePro Arg Arg Phe Ala Lys Val 1 5 10 15 Glu Thr Trp Gly Thr Ser Met MetAsp Leu Gly Val Gly Ser Phe 20 25 30 66 31 PRT Schizosaccharomyces pombehighly conserved GWT1 F-domain 66 Ile Leu Ala Val Asp Phe Thr Leu PhePro Arg Arg Tyr Ala Lys Val 1 5 10 15 Glu Thr Trp Gly Thr Ser Leu MetAsp Leu Gly Val Gly Ser Phe 20 25 30 67 17 PRT Saccharomyces cerevisiaehighly conserved GWT1 R-domain 67 Tyr Gln Glu His Val Thr Glu Tyr GlyVal His Trp Asn Phe Phe Ile 1 5 10 15 Thr 68 17 PRT Candida albicanshighly conserved GWT1 R-domain 68 Tyr Gln Glu His Glu Thr Glu Tyr GlyIle His Trp Asn Phe Phe Phe 1 5 10 15 Thr 69 17 PRT Schizosaccharomycespombe highly conserved GWT1 R-domain 69 Tyr Gln Glu His Val Ser Glu TyrGly Met His Trp Asn Phe Phe Phe 1 5 10 15 Thr

1. A DNA that encodes a protein having an activity to confer resistanceof a fungus against the compound shown in formula (Ia) when the DNA isoverexpressed in the fungus, wherein the DNA is selected from the groupconsisting of: (a) a DNA encoding a protein comprising the amino acidsequence of SEQ ID NO: 2, 4, 6, 28, 40, or 59, (b) a DNA comprising thenucleotide sequence of SEQ ID NO: 1, 3, 5, 27, 39, 41, 54, or 58, (c) aDNA that hybridizes under stringent conditions to a DNA comprising thenucleotide sequence of SEQ ID NO: 1, 3, 5, 27, 39, 41, 54, or 58, (d) aDNA encoding a protein comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 28, 40, or 59, wherein one or more amino acids have been added,deleted, substituted, and/or inserted, and (e) a DNA that is amplifiedusing SEQ ID NOS: 29 and 31 or SEQ ID NOS: 29 and 30 as primers


2. A DNA that encodes a protein having an activity to decrease theamount of a GPI-anchored protein in the cell wall of a fungus due to adefect in the function of the DNA, wherein the DNA is selected from thegroup consisting of: (a) a DNA encoding a protein comprising the aminoacid sequence of SEQ ID NO: 2, 4, 6, 28, 40, or 59, (b) a DNA comprisingthe nucleotide sequence of SEQ ID NO: 1, 3, 5, 27, 39, 41, 54, or 58,(c) a DNA that hybridizes under stringent conditions to a DNA comprisingthe nucleotide sequence of SEQ ID NO: 1, 3, 5, 27, 39, 41, 54, or 58,(d) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 2, 4, 6, 28, 40, or 59, wherein one or more amino acids have beenadded, deleted, substituted, and/or inserted, and (e) a DNA that isamplified using SEQ ID NOS: 29 and 31 or SEQ ID NOS: 29 and 30 asprimers.
 3. A protein encoded by the DNA of claim 1 or
 2. 4. A vectorinto which the DNA of claim 1 or 2 has been inserted.
 5. A transformantharboring the DNA of claim 1 or 2, or the vector of claim
 4. 6. Thetransformant of claim 5 which is a fungus that overexpresses the proteinof claim
 3. 7. A fungus, wherein the function of the protein of claim 3is defective.
 8. A method for producing the protein of claim 3, whichcomprises the steps of culturing the transformant of claim 5, andcollecting the expressed protein from the transformant, or from theculture supernatant thereof.
 9. An antibody that binds to the protein ofclaim
 3. 10. A method of screening for a compound having an antifungalaction, wherein the method comprises the steps of: (a) contacting a testsample with the protein of claim 3; (b) detecting the binding activitybetween the protein and the test sample; and (c) selecting a compoundhaving an activity to bind to the protein.
 11. A method of screening fora compound that has an antifungal action, which comprises the steps of:(a) contacting a test sample with a fungus that is overexpressing theprotein of claim 3; (b) detecting the amount of transport of aGPI-anchored protein to the cell wall in the fungus; and (c) selecting acompound that diminishes the amount of transport of the GPI-anchoredprotein to the cell wall detected in step (b) as compared to the amountof transport detected when the test sample was contacted with a fungusthat is not overexpressing the protein of claim
 3. 12. A compound havingan antifungal action that is isolated by the screening of claim 10 or11.
 13. An antifungal agent, comprising as an active ingredient acompound that inhibits the transport of GPI-anchored proteins to thecell wall of a fungus.
 14. An antifungal agent, comprising as an activeingredient the antibody of claim 9 or the compound of claim
 12. 15. Theantifungal agent of claim 13, comprising as an active ingredient thecompound represented by the general formula (I), a salt thereof, or ahydrate thereof, wherein in formula (I):

[R^(1a) and R^(2a) are identical to or different from each other anddenote individually a hydrogen atom, halogen atom, hydroxyl group, nitrogroup, cyano group, trifluoromethyl group, trifluoromethoxy group, asubstituted or unsubstituted C₁₋₆ alkyl group, C₂₋₆ alkenyl group, C₂₋₆alkynyl group, a substituted or unsubstituted C₁₋₆ alkoxy group, or agroup represented by the formula:

(wherein X¹ stands for a single bond, carbonyl group, or a grouprepresented by the formula —S(O)₂—; R^(5a) and R^(6a) are identical toor different from each other and denote a hydrogen atom or a substitutedor unsubstituted C₁₋₆ alkyl group); R^(1a) and R^(2a) may form togethera condensed ring selected from the group consisting of a substituted orunsubstituted benzene ring, a substituted or unsubstituted pyridinering, a substituted or unsubstituted pyrrole ring, a substituted orunsubstituted thiophene ring, a substituted or unsubstituted furan ring,a substituted or unsubstituted pyridazine ring, a substituted orunsubstituted pyrimidine ring, a substituted or unsubstituted pyrazinering, a substituted or unsubstituted imidazole ring, a substituted orunsubstituted oxazole ring, a substituted or unsubstituted thiazolering, a substituted or unsubstituted pyrazole ring, a substituted orunsubstituted isoxazole ring, a substituted or unsubstituted isothiazolering, a substituted or unsubstituted cyclohexane ring, and a substitutedor unsubstituted cyclopentane ring; R^(3a) and R^(4a) are identical toor different from each other and denote individually a hydrogen atom,halogen atom, hydroxyl group, nitro group, cyano group, carboxyl group,formyl group, hydroxyimino group, trifluoromethyl group,trifluoromethoxy group, C₁₋₆ alkyl group, C₁₋₆ alkoxy group, C₂₋₆alkenyl group, C₂₋₆ alkynyl group, a group represented by the formula—C(O)NR^(7a)R^(7b) (wherein R^(7a) and R^(7b) are identical to ordifferent from each other and denote individually a hydrogen atom, or aC₁₋₆ alkyl group), the formula —CO₂R^(7a) (wherein R^(7a) has the samemeaning as defined above), the formula —S(O)_(n)R^(7a) (wherein n standsfor an integer of 0 to 2 and R^(7a) has the same meaning as definedabove), the formula —S(O)₂NR^(7a)R^(7b) (wherein R^(7a) and R^(7b) havethe same meaning as defined above), a group of the formula

(wherein X² denotes a single bond, carbonyl group, or a group of theformula —S(O)₂—; R^(5b) and R^(6b) are identical to or different fromeach other, and denote a hydrogen atom, a substituted or unsubstitutedC₁₋₆ alkyl group, or a substituted or unsubstituted C₆₋₁₄ aryl group),or a group of the formula —Z¹—Z² (wherein Z¹ denotes a single bond,oxygen atom, vinylene group, or ethynylene group; Z² denotes a singlebond, or a C₁₋₆ alkyl group substituted or unsubstituted with 0 to 4substituents); R^(3a) and R^(4a) may together stand for a methylenedioxygroup or 1,2-ethylenedioxy group, alternatively, R^(3a) and R^(4a) maytogether stand for the formation of a condensed ring selected from agroup consisting of a substituted or unsubstituted benzene ring,substituted or unsubstituted pyridine ring, substituted or unsubstitutedpyrrole ring, substituted or unsubstituted thiophene ring, substitutedor unsubstituted furan ring, substituted or unsubstituted pyridazinering, substituted or unsubstituted pyrimidine ring, substituted orunsubstituted pyrazine ring, substituted or unsubstituted imidazolering, substituted or unsubstituted oxazole ring, substituted orunsubstituted thiazole ring, substituted or unsubstituted pyrazole ring,substituted or unsubstituted isoxazole ring, substituted orunsubstituted isothiazole ring, substituted or unsubstituted cyclohexanering, and substituted or unsubstituted cyclopentane ring, except incases where both R^(1a) and R^(2a) do not stand for hydrogen atoms]. 16.The antifungal agent of claim 13, comprising as the active ingredientcompound (Ia) of the formula:


17. A compound represented by the formula (II), a salt or a hydratethereof, wherein in formula (II),

[Ar stands for a substituent selected from a group consisting of theformulae (IIIa) to (IIIf):

(wherein K denotes a sulfur atom, oxygen atom, or a group represented bythe formula —NH—; R^(1b) and R_(2b) are identical to or different fromeach other and denote individually a hydrogen atom, halogen atom,hydroxyl group, nitro group, cyano group, trifluoromethyl group,trifluoromethoxy group, a group represented by the formula

(wherein X³ denotes a single bond, carbonyl group, or a grouprepresented by the formula —S(O)₂—; R^(5c) and R^(6c) are identical toor different from each other and denote a hydrogen atom, or asubstituted or unsubstituted C₁₋₆ alkyl group), or a group representedby the formula —X⁴—R^(8a) (wherein X⁴ denotes a single bond, oxygenatom, or sulfur atom; R^(8a) denotes a C₁₋₆ alkyl group, C₂₋₆ alkenylgroup, C₂₋₆ alkynyl group, C₃₋₈ cycloalkyl group, or C₃₋₈ cycloalkenylgroup); R^(1b) and R^(2b) together may form a methylenedioxy group, or a1,2-ethylenedioxy group); R^(3b) and R^(4b) are identical to ordifferent from each other and denote individually a hydrogen atom,halogen atom, hydroxyl group, nitro group, cyano group, carboxyl group,formyl group, hydroxyimino group, trifluoromethyl group,trifluoromethoxy group, C₁₋₆ alkyl group, C₁₋₆ alkoxy group, C₂₋₆alkenyl group, C₂₋₆ alkynyl group, or a group represented by the formula—Z^(1b)—Z^(2b) (wherein Z^(1b) denotes a single bond, vinylene group, orethynylene group; Z^(2b) denotes a single bond, or a C₁₋₆ alkyl groupthat is substituted or unsubstituted with 0 to 4 substituents); exceptin cases where (1) Ar stands for the aforementioned formula (IIId)wherein R^(1b) and R^(2b) are both hydrogen atoms, (2) at least one ofR³b or R⁴b denotes a hydrogen atom and the other is a hydrogen atom,methoxy group, hydroxyl group, methyl group, benzyloxy group, or ahalogen atom, and Ar stands for the aforementioned formula (IIIc)wherein R^(1b) and R^(2b) both denote hydrogen atoms or methoxy groups,(3) at least one of R^(3b) or R^(4b) denotes a hydrogen atom and theother is a hydrogen atom, hydroxyl group, methoxy group, or benzyloxygroup, and Ar stands for the formula (IIIc) wherein R^(1b) and R^(2b)both denote hydroxyl groups or benzyloxy groups, or (4) Ar stands forthe formula (IIId) wherein R^(1b) is a hydrogen atom and R^(2b) is aformyl group, hydroxymethyl group, or methoxycarbonyl group].
 18. Thecompound of claim 17, or a salt or hydrate thereof, wherein Ar standsfor the formula:

(wherein R^(1c) denotes a hydrogen atom, a substituted or unsubstitutedC₁₋₆ alkyl group, or a benzyl group), and excluding the case when R³bdenotes a hydrogen atom.
 19. A compound represented by the formula(IIIc2), or a salt or hydrate thereof, wherein in formula (IIIc2),

[R^(1b) and R^(2b) have the same meaning as defined above, except incases wherein (1) R^(1b) denotes a group represented by the formulaR^(1c)—O— (wherein R^(1c) has the same meaning as defined above), R^(2b)is a hydrogen atom, and R^(3b) denotes a hydrogen atom, (2) at least oneof R^(3b) or R^(4b) denotes a hydrogen atom, and the other is a hydrogenatom, methoxy group, hydroxyl group, methyl group, benzyloxy group, or ahalogen atom, and R^(1b) and R^(2b) both denote hydrogen atoms ormethoxy groups, or (3) at least one of R^(3b) or R^(4b) denotes ahydrogen atom, and the other is a hydrogen atom, hydroxyl group, methoxygroup, or benzyloxy group, and R^(1b) and R^(2b) both denote hydroxylgroups or benzyloxy groups].
 20. The antifungal agent of claim 17,having an antifungal action.
 21. The antifungal agent of claim 15,wherein at least one of R^(3a) and R^(4a) denotes a group represented bythe formula —C(O)NR^(7a)R^(7b) (wherein R^(7a) and R^(7b) have the samemeaning as defined above), the formula —CO₂R^(7a) (wherein R^(7a) hasthe same meaning as defined above), the formula —S(O)_(n)R^(7a) (whereinn denotes an integer of 0 to 2 and R^(7a) has the same meaning asdefined above), the formula —S(O)₂NR^(7a) R^(7b) (wherein R^(7a) andR^(7b) have the same meaning as defined above), the formula

(wherein X², R^(5b), and R^(6b) have the same meaning as defined above),or a C₁₋₆ alkoxy group substituted or unsubstituted with 0 to 4substituents, or R^(3a) and R^(4a) together denote a methylenedioxygroup, or a 1,2-ethylenedioxy group.
 22. The antifungal agent of claim15, wherein the compound having an antifungal action is (1)1-benzylisoquinoline, (2) 1-(4-bromobenzyl)isoquinoline, (3)1-(4-chlorobenzyl)isoquinoline, (4) 1-(4-fluorobenzyl)isoquinoline, (5)1-(4-iodobenzyl)isoquinoline, (6) 1-(3-methylbenzyl)isoquinoline, (7)1-(4-methylbenzyl)isoquinoline, (8) 1-(3,4-dimethylbenzyl)isoquinoline,(9) 1-(3-methoxybenzyl)isoquinoline, (10)1-(4-methoxybenzyl)isoquinoline, (11)1-(3,4-methylenedioxybenzyl)isoquinoline, (12)1-(4-benzyloxybenzyl)isoquinoline, (13) 1-(4-cyanobenzyl)isoquinoline,(14) 1-(4-nitrobenzyl)isoquinoline, (15) 1-(4-aminobenzyl)isoquinoline,(16) 1-(4-methoxybenzyl)-6,7-dichloro-isoquinoline, (17)1-(4-methoxy-2-nitro-benzyl)-isoquinoline, (18)1-(4-methoxybenzyl)-6,7-methylenedioxy-isoquinoline, (19)1-(2-amino-4-methoxy-benzyl)isoquinoline, (20)1-(4-methoxybenzyl)-7-hydroxy-6-methoxy-isoquinoline, (21)1-(4-benzyloxybenzyl)-6,7-dimethoxy-isoquinoline, (22)1-(4-methoxybenzyl)-6,7-dimethoxy-isoquinoline, (23)1(4-methoxy-2-nitro-benzyl)-isoquinoline, (24)3-(4-(1-isoquinolylmethyl)phenoxylpropylcyanide, (25)1-[4-(2,2,3,3-tetrafluoropropoxy)benzyl]isoquinoline, (26)1-[4-(2-piperidinoethoxy)benzyl]isoquinoline, (27)4-(1-isoquinolylmethyl)phenyl(2-morpholinoethyl)ether, (28)1-[4-(2-methoxyethoxy)benzyl]isoquinoline, (29)N-{2-[4-(1-isoquinolylmethyl)phenoxy]ethyl}-N,N-dimethylamine, (30)1-[4-(phenethyloxy)benzyl]isoquinoline, (31)1-{4-[(2-methylallyl)oxy]benzyl}isoquinoline, (32)1-(4-isobutoxybenzyl)isoquinoline, (33)1-[4-(2-phenoxyethoxy)benzyl]isoquinoline, (34) methyl2-[4-(1-isoquinolylmethyl)phenoxy]acetate, (35)2-[4-(1-isoquinolylmethyl)phenoxy]-1-ethanol, (36) t-butylN-{2-[4-(1-isoquinolylmethyl)phenoxy]ethyl}carbamate, (37)1-{4-[3-(tetrahydro-2H-2-pyranyloxy)propoxy]benzyl}isoquinoline, (38)2-[4-(1-isoquinolylmethyl)phenoxy]-1-ethaneamine, (39)1-[4-(3-piperidinopropoxy)benzyl]isoquinoline, (40)3-[4-(1-isoquinolylmethyl)phenoxy]-1-propanol, (41)1-[4-(2-ethylbutoxy)benzyl]isoquinoline, (42)4-[4-(1-isoquinolylmethyl)phenoxy]butanoic acid, (43)1-(4-{3-[(4-benzylpiperazino)sulfonyl]propoxy}benzyl)isoquinoline, (44)1-(4-{3-[4-(4-chlorophenyl)piperazino]propoxy}benzyl)isoquinoline, (45)4-(1-isoquinolylmethyl)aniline, (46)N-(4-(1-isoquinolylmethyl)phenyl]butaneamide, (47)N-[4-(1-isoquinolylmethyl)phenyl]propaneamide, (48)N-[4-(1-isoquinolylmethyl)phenyl]-1-ethanesulfonamide, (49)N-[4-(1-isoquinolylmethyl)phenyl]-N-methyl-ethanesulfonamide, (50)N-[4-(1-isoquinolylmethyl)phenyl]-N-methylamine, (51)N-[4-(1-isoquinolylmethyl)phenyl]-N-propylamine, or (52)N-[4-(1-isoquinolylmethyl)phenyl]-N-methyl-N-propylamine.
 23. A methodfor treating a mycotic infection comprising administering atherapeutically effective dose of any one of the antifungal agents ofclaims 13 to 22 to a mammal.